Method for high-throughput screening of compounds and combinations of compounds for discovery and quantification of actions, particularly unanticipated therapeutic or toxic actions, in biological systems

ABSTRACT

The invention enables high-throughput screening of compounds in living systems to detect unanticipated or unintended biological actions. The invention also allows for screening, detection, and confirmation of new indications for approved drugs. Screening and detection of toxic effects of compounds also can be achieved by using the methods of the invention. The methods comprise administering isotope-labeled substrates to a living system so that the label is incorporated into molecules in a manner that reveals flux rates through metabolic pathways thought to be involved in a disease. Comparisons between living systems exposed to compounds and living systems not so exposed reveals the effects of the compounds on the flux rates through the metabolic pathways. Combinations or mixtures of compounds can be systematically screened to detect unantidpated or unintended biological actions, including synergistic actions, in the same manner.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation patent application of U.S. patentapplication Ser. No. 10/997,323, with a filing date of Nov. 23, 2004,and claims the benefit of U.S. Provisional Patent Application Ser. No.60/525,261 filed Nov. 25, 2003, which are incorporated herein byreference in their entirety.

FIELD OF THE INVENTION

The invention relates to methods for screening compounds, combinationsof compounds, or mixtures of compounds (i.e., drugs and drug candidates,including chemical entities, whether new or known, and biologicalfactors, whether new or known) for actions in biological systems. Thedisclosed methods measure and quantify molecular flux rates throughmetabolic pathways (synthesis and breakdown or input and removal ratesfrom pools of molecules) in vivo as targets of drug action. Thedisclosed methods are capable of high-throughput, large-scale, automatedapplications. The methods are particularly applicable to detecting andestablishing unanticipated or unintended actions of drugs or drugcandidates during drug discovery, development and approval (DDDA). Themethods disclosed herein are particularly suitable for establishingsecondary therapeutic claims (“new uses” or “new indications”) anddetermining toxicities both of known compounds and new compounds.

BACKGROUND OF THE INVENTION

The contemporary system of drug discovery, development and approval(DDDA) is highly specific and target-directed. The system is built on amodel of intended biochemical and molecular actions. New compounds(i.e., chemical entities or biological factors) are identified,optimized and evaluated based on their actions on intended therapeutictargets. These intended therapeutic targets are typically proteins orgenes believed to be involved in a disease process of interest (the drugtarget). Increasingly, other cellular macromolecules are also becomingimportant as drug targets (e.g., mRNA).

Actions on the drug target are evaluated against large numbers ofcompounds by use of high-throughput screening (HTS) assays that measurethe activity or state of the protein or gene target. Compounds showingpotentially useful activity on the drug target are termed lead compounds(also known as “drug leads”). Once identified, drug leads are filteredand selected on the basis of their activity on the disease processtargeted and, ultimately, on clinical end-points. FDA approval isultimately given for single, well-defined clinical indications that areidentified and defined and tested in advance in specified diseases.

Drug leads are therefore both discovered and developed in the context ofa highly constrained set of protocols built on a model of intendedactions. Drug targets are specific and are identified in advance fordiscovery initiatives. Stated differently, FDA approval of a drug leadis not obtained by administration of the compound to diverse people witha variety of random medical disorders to see if it helps one or more ofthese, but occurs within an explicit context of prospectively definedeffects in specific disease states.

This approach of contemporary DDDA has major flaws. First, identifying“hits” by HTS assays against molecular targets hypothesized to beinvolved in a disease does not in fact establish or prove activityagainst the disease. Activity against disease still has to be validatedindependently. Indeed, true in vivo activity and efficacy of drug leadsmay be unreliably or misleadingly assessed by HTS molecular assays.Second, subsequent validation of drug leads against physiologic modelsof disease often remains highly inefficient for the chronic conditionsthat are the major therapeutic targets of current drug research, therebyleading to a downstream roadblock in the DDDA system in the filteringsteps. Third, unintended toxic actions of drugs are not identified bythis approach. Fourth, unintended or secondary therapeutic actions(i.e., other actions besides the effects on the specific moleculartarget screened) also are not identified efficiently by this approach,thereby missing out on the detection and discovery of other potentialtherapeutic uses of compounds on which a pharmaceutical company isalready investing time and money to develop. Fifth, synergistic effectsof combination therapies on a disease process, occurring by interactionsamong compounds that act on different biochemical steps in the diseasepathway, are not detectable by screening approaches which only measureone step in a disease pathway at a time.

The ideal solution to the problem of unintended actions and functionalimportance of compounds discovered through screening for specific,intended molecular actions is therefore evident: a systematic method formeasuring and identifying unintended actions and functional consequencesof compounds, combinations of compounds, and mixtures of compounds. Theavailability of a systematic procedure for efficient, high-throughputdiscovery and confirmation of unintended actions of compounds orcombinations of compounds or mixtures of compounds on functionallyrelevant biological processes would therefore radically alter the entireDDDA process. Such methods are disclosed herein, in addition to methodsfor screening and comparing compounds and combinations of compounds andmixtures of compounds for actions on intended processes.

SUMMARY OF THE INVENTION

The present invention is directed to methods for measuring andquantifying molecular flux rates within one or more metabolic pathwaysof interest, in response to exposure to one or more compounds,combinations of compounds, or mixtures of compounds, the methodsenabling an investigator to discover an unanticipated or unexpectedaction or actions (or both) elicited by the one or more compounds,combination of compounds, or mixtures of compounds. The methods of theinvention include high-throughput screening assays. In one embodiment ofthe invention, the unanticipated or unexpected action is a therapeuticaction. In another embodiment of the invention, the unanticipated orunexpected action is a toxic effect. The methods of the invention permitthe identification of compounds that alter the metabolic flux rates ofonce or more metabolic pathways.

In the present invention, one or more isotope-labeled substrates (i.e.,metabolic precursors) are administered to a cell, tissue or organism fora period of time sufficient for the isotope label to be incorporatedinto one or more targeted molecules in at least one targeted metabolicpathway of interest. More than one isotope label may be administered.Measurement of the isotopic content and/or pattern or the rate of changeof the isotopic content and/or pattern in the one or more targetedmolecules is performed to calculate the molecular flux rate in the oneor more pathways of interest. One or more compounds are administered(constituting exposure) to a living system and the molecular flux ratesare measured in the presence and absence of exposure. In a furtherembodiment of the invention, the molecular flux rates are measured inresponse to a specific dose or a range of doses of the one or morecompounds. In another embodiment of the invention, the molecular fluxrates are measured in response to exposure to a combination ofcompounds. In still another embodiment of the invention, the molecularflux rates are measured in response to exposure to a mixture ofcompounds.

The one or more compounds can be a new chemical entity, a chemicalentity drug lead (i.e., a “small molecule” drug lead), or a knownchemical entity drug (i.e., a “small molecule” drug), for example analready-approved drug listed in the Physician's Desk Reference (PDR) orthe Merck Index (e.g., a drug approved by the FDA or other correspondingagencies outside the U.S.). The one or more compounds also can be a newbiological factor or a known biological factor including analready-approved biological factor drug. The one or more compounds canbe selected randomly or on the basis of a specific biochemical rationaleconcerning a hypothesized role in the molecular pathogenesis of one ormore diseases.

The invention allows for the comparison between the molecular flux ratesmeasured from exposed cells, tissues, or organisms of the living systemto the molecular flux rates measured from non-exposed cells, tissues, ororganisms of the living system. Differences between the exposed andnon-exposed molecular flux rates are identified and this information isthen used to determine whether one or more compounds (or combinations ofcompounds or mixtures of compounds) elicit a metabolic action on the oneor more pathways of interest on the exposed cell, tissue, or organism.The metabolic action of the compound (or combination of compounds ormixture of compounds) on the exposed cell, tissue or organism may beunexpected or unanticipated (based on prevailing biochemical knowledgeand concepts about the compound and molecular flux within the pathway)or may be anticipated or expected. The one or more compounds (orcombination of compounds or mixture of compounds) can be administered toa mammal and the molecular flux rates calculated and evaluated againstthe molecular flux rates calculated from an unexposed mammal of the samespecies. The mammal may be a human.

In another embodiment of the invention, the molecular flux rates aremeasured in one or more metabolic pathways involved in the molecularpathogenesis of a disease. In a further embodiment, the one or moremetabolic pathways are the cause of the disease or contribute to theinitiation, progression, activity, pathologic consequences, symptoms, orseverity of the disease.

In another embodiment of the invention, the molecular flux rates fromone or more metabolic pathways are measured concurrently. In a furtherembodiment, the molecular flux rates are measured using stableisotope-labeling techniques. The stable isotope label may includespecific heavy isotopes of elements present in biomolecules, such as ²H,¹³C, ¹⁵N, ¹⁸O, ³³S, ³⁴S. In another embodiment, the molecular flux ratesare measured using radioactive isotope-labeling techniques. Theradioactive isotope label may include ³H, ¹⁴C, ³⁵S, ¹²⁵I, ¹³¹I.

Isotope-labeled precursors include, but are not limited to, ²H₂O, H₂¹⁸O, ³H₂O, ¹⁵NH₃, ¹³CO₂, H¹³CO₃, ²H-labeled amino acids, ¹³C-labeledamino acids, ¹⁵N-labeled amino acids, ¹⁸O-labeled amino acids, ³⁴S or³³S-labeled amino acids, ³H-labeled amino acids, and ¹⁴C-labeled aminoacids.

The isotope substrate may be chosen from ²H₂O, ²H-glucose, ²H-labeledamino acids, ¹³C-labeled amino acids, ²H-labeled organic molecules,¹³C-labeled organic molecules, and ¹⁵N-labeled organic molecules labeledwater. The isotope substrate may be labeled water, for example, ²H₂O, H₂¹⁸O, or ³H₂O. The labeled water may be ²H₂O.

Stable isotope-labeled substrates are incorporated into one or moremolecules of one or more metabolic pathways of interest. In this manner,the molecular flux rates can be determined by measuring, over specifictime intervals, isotopic content and/or pattern or rate of change ofisotopic content and/or pattern in the targeted molecules, for exampleby using mass spectrometry, allowing for the determination of themolecular flux rates within the one or more metabolic pathways ofinterest, by use of analytic and calculation methods known in the art.

Alternatively, the use of radiolabeled substrates is contemplated foruse in the present invention wherein the radiolabeled substrates areincorporated into one or more molecules of one or more metabolicpathways of interest. In this manner, the molecular flux rates can bedetermined by measuring radiation and/or radioactivity of the targetedmolecules of interest within the one or more metabolic pathways ofinterest by using techniques well known in the art such as scintillationcounting. The molecular flux rates within the one or more metabolicpathways of interest are then calculated, using methods known in theart.

In another embodiment of the invention, isolated isotopically perturbedmolecules are provided, the isotopically perturbed molecules includingone or more isotopes. The isolated isotopically perturbed molecules areproducts of the labeling methods described herein. The isolatedisotopically perturbed molecules are collected by sampling techniquesknown in the art and are analyzed using appropriate analytical tools. Inone embodiment of the invention, the isolated isotopically perturbedmolecules are comprised of one or more stable isotopes. In anotherembodiment, the isolated isotopically perturbed molecules are comprisedof one or more radioactive isotopes.

In yet another embodiment of the invention, one or more kits areprovided that comprise isotope-labeled precursors and instructions forusing them. The kits may contain stable-isotope labeled precursors orradioactive-labeled isotope precursors or both. Stable-isotope labeledprecursors and radioactive-labeled isotope precursors may be provided inone kit or they may be separated and provided in two or more kits. Thekits may further comprise one or more tools for administering theisotope-labeled precursors. The kits also may comprise one or more toolsfor collecting samples from a subject.

In yet another embodiment of the invention, one or more informationstorage devices are provided that comprise data generated from themethods of the present invention. The data may be analyzed, partiallyanalyzed, or unanalyzed. The data may be imprinted onto paper, plastic,magnetic, optical, or other medium for storage and display.

In yet another embodiment of the invention, one or more compounds,combinations of compounds, or mixtures of compounds identified and atleast partially characterized by the methods of the present inventionare contemplated.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic diagram illustrating the contemporary model ofdrug discovery (testing for an intended action) versus a strategy oftesting for unintended actions.

FIG. 2 shows a schematic diagram illustrating the method of screeningfor new indications of approved drugs.

FIG. 3 shows a schematic diagram of an example metabolic pathway (DNAsynthesis, both de novo and salvage) and various component elements.Locations of stable or radioactive isotope labeling are shown.G6P=Glucose-6-phosphate. R5P=ribose-5-phosphate.PRPP=5-phosphoribosyl-α-pyrophospate. NDP=nucleotide diphosphate.dNTP=deoxynucleotide triphosphate. RR=ribonucleotide reductase.dN=deoxynucleotide. ³H-dT=tritiated deoxythymidine.BrdU=5-bromo-2-deoxyuridine. GNG=gluconeogenesis. DNNS=de novonucleotide synthesis. DNPS=de novo precursor synthesis.

FIG. 4 depicts the fractional replacement (turnover) of murine neuronsin vivo. Neurons were isolated from mice concurrently treated withintraperitoneal lipopolysaccharide (LPS) a known neuroinflammatorystimulus, and deuterated water for 45 days. Fractional replacement ofneurons was determined by GC/MS analysis of DNA from isolated neurons.Both doses of LPS resulted in a statistically significant increase inneuron turnover with respect to control (p<0.05), and statisticallysignificant dose dependence was also observed (p<0.05).

FIG. 5 shows a decrease in adipocyte proliferation (i.e., fat cellproliferation) in ob/ob mice after treatment with leptin.

FIG. 6 shows mouse liver cell proliferation during griseofulvinadministration (a=control, b=0.1% dose, c=0.2% dose, and d=0.5% dose, 5days exposure).

FIG. 7 depicts a decrease in mammary epithelial cell proliferation inboth intact female rats and ovariectomized rats after treatment withvery low doses of selective estrogen receptor modulators (tamoxifen andraloxifene).

FIG. 8 a shows that the addition of rosiglitazone to a high carbohydrate(HC) diet significantly increased n in the epididymal and inguinaladipose depots after 26 days of diet and 15 days of ²H₂O. FIG. 8 b showsthat by 75 days of diet, n was significantly higher in all adiposedepots in the rosiglitazone group.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to methods of detecting actions,particularly unintended or unanticipated actions, of compounds,combinations of compounds, or mixtures of compounds (e.g., new chemicalentities, known drug agents including already-approved small molecularentities, new biological factors, or known biological factors includingalready-approved biological factors), in living systems by measuringmolecular flux rates (i.e., synthesis and breakdown or input and removalrates) within metabolic pathways of interest. Molecular flux rateswithin targeted metabolic pathways are used as biomarkers forestablishing and quantifying the actions of compounds. Anisotope-labeled substrate molecule is administered or contacted to oneor more cells, tissues, or organisms of a living system (i.e., exposingthe one or more cells, tissues, or organisms to the isotope-labeledsubstrate molecule, thereby constituting exposure to said one or morecells, tissues, or organisms). The isotopic content and/or pattern orrate of change of isotopic content and/or pattern of one or moretargeted molecules within one or more metabolic pathways of interest isthen measured, optimally by use of mass spectrometry, to determinemolecular flux rates within the one or more metabolic pathways ofinterest.

A compound, combination of compounds, or mixture of compounds is alsoadministered or contacted to one or more cells, tissues, or organisms.The molecular flux rates through one or more pathways of interest in theone or more cells, tissues, or organisms are then measured and comparedwith molecular flux rates through the pathways in one or more cells,tissues, or organisms not exposed to the compound, combination ofcompounds, or mixture of compounds. Combinations or mixtures ofcompounds including combinations or mixtures of known drug agents(including small molecule agents and biological factors) are tested fortheir effects on molecular flux rates through pathways of interest, toidentify and quantify synergistic or antagonistic actions of specificcombinations. In this manner, a systematic, high-throughput method foridentifying unintended actions or confirming biological importance ofcompounds or combinations of compounds is provided thereby overcomingcurrent limitations of DDDA that only measure a single, intendedmolecular action of a compound. Thus, the Applicant has discoveredmethods allowing for drug effects (unanticipated or unintended orconfirming biological importance) to be measured on any fully assembledbiological system (i.e., the ability to measure molecular flux rates inmultiple metabolic pathways, both concurrently and independently, inliving systems).

The methods of the present invention allow for high-throughput screeningof compounds, combinations of compounds, or mixtures of compoundsthereby allowing for the systematic discovery of secondary orunanticipated actions of entire classes of therapeutic agents that weredeveloped or approved for other actions (i.e., thereby providing amethod for “mining” the Physicians Desk Reference or Merck Index for newuses of known drugs or agents, a process that is referred to as “drugrepositioning” or “drug repurposing”). The methods of the presentinvention also allow for the discovery of unanticipated toxic effects ofcompounds, combinations of compounds, or mixtures of compounds.

The invention has applications in drug discovery, development andapproval as well as in subsequent medical diagnostics, clinicalmanagement of patients and disease prevention.

I. General Techniques

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry andimmunology, which are within the skill of the art. Such techniques areexplained fully in the literature, such as, Molecular Cloning: ALaboratory Manual, second edition (Sambrook et al., 1989) Cold SpringHarbor Press; Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Methodsin Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook(J. E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R. I.Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J. P.Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture:Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell,eds., 1993-8) J. Wiley and Sons; Methods in Enzymology (Academic Press,Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C.Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M.Miller and M. P. Cabs, eds., 1987); Current Protocols in MolecularBiology (F. M. Ausubel et al., eds., 1987); PCR: The Polymerase ChainReaction, (Mullis et al., eds., 1994); Current Protocols in Immunology(J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology(Wiley and Sons, 1999); and Mass isotopomer distribution analysis ateight years: theoretical, analytic and experimental considerations byHellerstein and Neese (Am J Physiol 276 (Endocrinol Metab. 39)E1145-E1162, 1999). Furthermore, procedures employing commerciallyavailable assay kits and reagents will typically be used according tomanufacturer-defined protocols unless otherwise noted.

II. Definitions

Unless otherwise defined, all terms of art, notations and otherscientific terminology used herein are intended to have the meaningscommonly understood by those of skill in the art to which this inventionpertains. In some cases, terms with commonly understood meanings aredefined herein for clarity and/or for ready reference, and the inclusionof such definitions herein should not necessarily be construed torepresent a substantial difference over what is generally understood inthe art. The techniques and procedures described or referenced hereinare generally well understood and commonly employed using conventionalmethodology by those skilled in the art, such as, for example, Massisotopomer distribution analysis at eight years: theoretical, analyticand experimental considerations by Hellerstein and Neese (Am J Physiol276 (Endocrinol Metab. 39) E1146-E1162, 1999). As appropriate,procedures involving the use of commercially available kits and reagentsare generally carried out in accordance with manufacturer definedprotocols and/or parameters unless otherwise noted.

“Molecular flux rates” refers to the rate of synthesis and/or breakdownof molecules within a cell, tissue, or organism. “Molecular flux rates”also refers to a molecule's input into or removal from a pool ofmolecules, and is therefore synonymous with the flow into and out ofsaid pool of molecules.

“Metabolic pathway” refers to any linked series of two or morebiochemical steps in a living system, the net result of which is achemical, spatial or physical transformation of a molecule or molecules.Metabolic pathways are defined by the direction and flow of moleculesthrough the biochemical steps that comprise the pathway. Moleculeswithin metabolic pathways can be of any biochemical class, e.g.,including but not limited to lipids, proteins, amino acids,carbohydrates, nucleic acids, polynucleotides, porphyrins,glycosaminoglycans, glycolipids, intermediary metabolites, inorganicminerals, ions, etc.

“Flux rate through a metabolic pathway” refers to the rate of moleculartransformations through a defined metabolic pathway. The unit of fluxrates through pathways is chemical mass per time (e.g., moles perminute, grams per hour). Flux rate through a metabolic pathway optimallyrefers to the transformation rate from a clearly defined biochemicalstarting point to a clearly defined biochemical end-point, including allthe stages in between in the defined metabolic pathway of interest.

“Isotopes” refer to atoms with the same number of protons and hence ofthe same element but with different numbers of neutrons (e.g., ¹H vs. ²Hor D).

“Isotopologues” refer to isotopic homologues or molecular species thathave identical elemental and chemical compositions but differ inisotopic content (e.g., CH₃NH₂ vs. CH₃NHD in the example above).Isotopologues are defined by their isotopic composition, therefore eachisotopologue has a unique exact mass but may not have a uniquestructure. An isotopologue is usually comprised of a family of isotopicisomers (isotopomers) which differ by the location of the isotopes onthe molecule (e.g., CH₃NHD and CH₂DNH₂ are the same isotopologue but aredifferent isotopomers).

“Isotope-labeled water” or “heavy water” includes water labeled with oneor more specific heavy isotopes of either hydrogen or oxygen. Specificexamples of isotope-labeled water include ²H₂O, ³H₂O, and H₂ ¹⁸O.

“Action” includes any biological process or event induced in a livingsystem by a compound.

“Unanticipated or unintended action” includes any biological process orevent induced in a living system by a compound that was not previouslyused as an outcome measure in the design or development of the compound(e.g., in a high-throughput screening assay of an enzyme target, in acomputer-simulated model of an enzyme's active site, or in an in vivophysiologic model of an altered process); and therefore has not beenexplicitly predicted on a rational basis, from compelling biochemicalevidence, and is not taught as an action to expect from exposure to aparticular compound.

“Compound” is used herein to describe any composition of matterincluding a chemical entity or a biological factor that is administered,approved or under testing as potential therapeutic agent or is a knowntherapeutic agent. Thus the term encompasses chemical entities andbiological factors as defined, infra.

“Chemical entity” includes any chemical, whether new (i.e., a “newchemical entity” or NCE) or known (e.g., a small molecule drug lead orsmall molecule already-approved drug), that is administered to one ormore cells, tissues, or organisms for the purpose of screening it forbiological or biochemical activity toward the goal of discovering itsuse as a potential therapeutic agent[s] (drug[s])).

“Biological factor” as used herein means any compound made by a livingsystem that is administered to one or more cells, tissues, or organismsfor the purpose of screening it for biological or biochemical activitytoward the goal of discovering its use as a potential therapeuticagent[s] (drug[s])). Examples of biological factors include, but are notlimited to, antibodies, hormones, enzymes, enzyme cofactors, peptides,secreted proteins, intracellular proteins, membrane-bound proteins,lipids, phospholipids, carbohydrates, fatty acids, amino acids, nucleicacids (including deoxyribonucleic acids and ribonucleic acids),steroids, and the like. Biological factors also include those compoundsmade by a living system that have been subsequently altered, modified,or optimized, for example, by way of laboratory techniques.

“Drug leads” or “drug candidates” are herein defined as compounds thatare being evaluated, either preclinically or clinically, as potentialtherapeutic agents (drugs).

“Known drugs” or “known agents” refers to compounds that have beenapproved for therapeutic use as drugs in human beings or animals in theUnited States.

By “high-throughput screening” is meant the rapid and efficientscreening of large numbers of compounds for potential actions.

“Living system” includes, but is not limited to, cells, cell lines,tissues, animal models of disease, guinea pigs, rabbits, dogs, cats,other pet animals, mice, rats, non-human primates, and humans.

A “biological sample” encompasses any sample obtained from a cell,tissue, organism, or individual. The definition encompasses blood andother liquid samples of biological origin, that are accessible from anorganism through sampling by invasive means (e.g., surgery, open biopsy,endoscopic biopsy, and other procedures involving non-negligible risk)or by minimally invasive or non-invasive approaches (e.g., urinecollection, blood drawing, needle aspiration, and other proceduresinvolving minimal risk, discomfort or effort). The definition alsoincludes samples that have been manipulated in any way after theirprocurement, such as by treatment with reagents, solubilization, orenrichment for certain components, such as proteins or organicmetabolites. The term “biological sample” also encompasses a clinicalsample such as serum, plasma, other biological fluid, or tissue samples,and also includes cells in culture, cell supernatants and cell lysates.

“Biological fluid” refers, but is not limited to, urine, blood,interstitial fluid, edema fluid, saliva, lacrimal fluid, inflammatoryexudates, synovial fluid, abscess, empyema or other infected fluid,cerebrospinal fluid, sweat, pulmonary secretions (sputum), seminalfluid, feces, bile, intestinal secretions, or other biological fluid.

“Exact mass” refers to mass calculated by summing the exact masses ofall the isotopes in the formula of a molecule (e.g., 32.04847 forCH₃NHD).

“Nominal mass” refers to the integer mass obtained by rounding the exactmass of a molecule.

“Mass isotopomer” refers to family of isotopic isomers that is groupedon the basis of nominal mass rather than isotopic composition. A massisotopomer may comprise molecules of different isotopic compositions,unlike an isotopologue (e.g., CH₃NHD, ¹³CH₃NH₂, CH₃ ¹⁵NH₂ are part ofthe same mass isotopomer but are different isotopologues). Inoperational terms, a mass isotopomer is a family of isotopologues thatare not resolved by a mass spectrometer. For quadrupole massspectrometers, this typically means that mass isotopomers are familiesof isotopologues that share a nominal mass. Thus, the isotopologuesCH₃NH₂ and CH₃NHD differ in nominal mass and are distinguished as beingdifferent mass isotopomers, but the isotopologues CH₃NHD, CH₂DNH₂,¹³CH₃NH₂, and CH₃ ¹⁵NH₂ are all of the same nominal mass and hence arethe same mass isotopomers. Each mass isotopomer is therefore typicallycomposed of more than one isotopologue and has more than one exact mass.The distinction between isotopologues and mass isotopomers is useful inpractice because all individual isotopologues are not resolved usingquadrupole mass spectrometers and may not be resolved even using massspectrometers that produce higher mass resolution, so that calculationsfrom mass spectrometric data must be performed on the abundances of massisotopomers rather than isotopologues. The mass isotopomer lowest inmass is represented as M₀; for most organic molecules, this is thespecies containing all ¹²C, ¹H, ¹⁶O, ¹⁴N, etc. Other mass isotopomersare distinguished by their mass differences from M₀ (M₁, M₂, etc.). Fora given mass isotopomer, the location or position of isotopes within themolecule is not specified and may vary (i.e., “positional isotopomers”are not distinguished).

“Mass isotopomer envelope” refers to the set of mass isotopomerscomprising the family associated with each molecule or ion fragmentmonitored.

“Isotopic content” refers to the content of isotopes in a molecule orpopulation of molecules relative to the content in the molecule orpopulation of molecules naturally (i.e., prior to administration orcontacting of isotope labeled precursor subunits). The term “isotopeenrichment” is used interchangeably with isotopic content herein.

“Isotopic pattern” refers to the internal relationships of isotopiclabels within a molecule or population of molecules, e.g., the relativeproportions of molecular species with different isotopic content, therelative proportions of molecules with isotopic labels in differentchemical loci within the molecular structure, or other aspects of theinternal pattern rather than absolute content of isotopes in themolecule.

“Mass isotopomer pattern” refers to a histogram of the abundances of themass isotopomers of a molecule. Traditionally, the pattern is presentedas percent relative abundances where all of the abundances arenormalized to that of the most abundant mass isotopomer; the mostabundant isotopomer is said to be 100%. The preferred form forapplications involving probability analysis, such as mass isotopomerdistribution analysis (MIDA), however, is proportion or fractionalabundance, where the fraction that each species contributes to the totalabundance is used. The term “isotope pattern” may be used synonomouslywith the term “mass isotopomer pattern.”

“Monoisotopic mass” refers to the exact mass of the molecular speciesthat contains all ¹H, ¹²C, ¹⁴N, ¹⁶O, ³²S, etc. For isotopologuescomposed of C, H, N, O, P, S, F, Cl, Br, and I, the isotopic compositionof the isotopologue with the lowest mass is unique and unambiguousbecause the most abundant isotopes of these elements are also the lowestin mass. The monoisotopic mass is abbreviated as m₀ and the masses ofother mass isotopomers are identified by their mass differences from m₀(m₁, m₂, etc.).

“Isotopically perturbed” refers to the state of an element or moleculethat results from the explicit incorporation of an element or moleculewith a distribution of isotopes that differs from the distribution thatis most commonly found in nature, whether a naturally less abundantisotope is present in excess (enriched) or in deficit (depleted).

By “molecule of interest” is meant any molecule (polymer and/ormonomer), including but not limited to, amino acids, carbohydrates,fatty acids, peptides, sugars, lipids, nucleic acids, polynucleotides,glycosaminoglycans, polypeptides, or proteins that are present within ametabolic pathway within a living system.

“Monomer” refers to a chemical unit that combines during the synthesisof a polymer and which is present two or more times in the polymer.

“Polymer” refers to a molecule synthesized from and containing two ormore repeats of a monomer.

“Protein” refers to a polymer of amino acids. As used herein, a“protein” may refer to long amino acid polymers as well as shortpolymers such as peptides.

By “amino acid” is meant any amphoteric organic acid containing theamino group (i.e., NH₂). The term encompasses the twenty common (oftenreferred in the art as “standard” or sometimes as “naturally occurring”)amino acids as well as the less common (often referred in the art as“nonstandard”) amino acids. Examples of the twenty common amino acidsinclude the alpha-amino acids (or α-amino acids), which have the aminogroup in the alpha position, and generally have the formulaRCH—(NH₂)—COOH. The α-amino acids are the monomeric building blocks ofproteins and can be obtained from proteins through hydrolysis. Examplesof nonstandard amino acids include, but are not limited to raminobutyricacid, dopamine, histamine, thyroxine, citrulline, ornithine,homocysteine, and S-adenosylmethionine.

“Lipid” refers to any of a heterogeneous group of fats and fatlikesubstances characterized by being water insoluble and being extractableby nonpolar (or organic) solvents such as alcohol, ether, chloroform,benzene, etc. All contain as a major constituent aliphatic hydrocarbons.The lipids, which are easily stored in the body, serve as a source offuel, are an important constituent of cell structure, and serve otherbiological functions. Lipids include, but are not limited to fattyacids, neutral fats (e.g., triacylglycerols), waxes and steroids (e.g.,cholesterol). Complex lipids comprise the glycolipids, lipoproteins andphospholipids.

“Fatty acids” are carboxylic acids with long-chain hydrocarbon sidegroups. They are comprised of organic, monobasic acids, which arederived from hydrocarbons by the equivalent of oxidation of a methylgroup to an alcohol, aldehyde, and then acid. Fatty acids can be eithersaturated or unsaturated.

By “DNA” is meant a polymeric form of deoxyribonucleotides (adenine,guanine, thymine, or cytosine) in double-stranded or single-strandedform, either relaxed or supercoiled. This term refers only to theprimary and secondary structure of the molecule, and does not limit itto any particular tertiary forms. Thus, this term includes single- anddouble-stranded DNA found, inter alia, in linear DNA molecules (e.g.,restriction fragments), viruses, plasmids, and chromosomes. The termcaptures molecules that include the four bases adenine, guanine,thymine, or cytosine, as well as molecules that include base analogswhich are known in the art.

A “nucleic acid” sequence refers to a DNA or RNA sequence. The termcaptures sequences that include any of the known base analogues of DNAand RNA such as, but not limited to 4-acetylcytosine,8-hydroxy-N⁶-methyladenosine, aziridinylcytosine, pseudoisocytosine,5-(carboxyhydroxylmethyl) uracil, 5-fluorouracil, 5-bromouracil,5-carboxymethylaminomethyl-2-thiouracil,5-carboxymethyl-aminomethyluracil, dihydrouracil, inosine,N⁶-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N⁶-methyladenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5-methoxycarbonylmethyluracil, 5-methoxyuracil,2-methylthio-N⁶-isopentenyladenine, uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine,2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,5-methyluracil, N-uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and2,6-diaminopurine.

By “carbohydrate” is meant any compound of carbon, oxygen and hydrogen,of general formula Cx(H₂O)y, including sugars (monosaccharides anddisaccharides) and their derivatives, and polysaccharides such as starchand cellulose.

By “sugar” is meant the common name for any sweet, crystalline, simplecarbohydrate that is an aldehyde or ketone derivative of a polyhydricalcohol. Sugars are mainly disaccharides like sucrose andmonosaccharides like fructose or glucose; all are soluble in dilutealcohol or water and are white in their pure form. The term encompassesmonosaccharides, disaccharides, trisaccharides, heterosaccharides, orpolysaccharides (which are comprised of monosaccharide residues).Monosaccharides include glucose (both D-glucose and L-glucose), mannose,fructose galactose and sugar derivatives including, but not limited toN-acetylmuramic acid, N-acetylneuraminic acid and other sialic acids,N-acetylmannosamine, glucuronic acid, glucosamine, etc. Polysaccharidesinclude disaccharides such as sucrose, maltose and lactose and longerchain sugar molecules such as starch, glycogen, cellulose, chitin, etc.By the term “oligosaccharide” is meant a molecule comprised of a fewcovalently linked monosaccharide monomers.

By “glycosaminoglycan” is meant a polymer comprised of a network oflong, unbranched chains made up of repeating units of disaccharides thatcontain amino group sugars, at least one of which has a negativelycharged side group (carboxylate or sulfate). Examples ofglycosaminoglycans include, but are not limited to hyaluronate(D-glucuronic acid-N-acetyl-D-glucosamine: MW up to 10 million),chondroitin sulfate (D-glucuronic acid-N-acetyl-D-galactosamine-4 or6-sulfate), dermatan sulfate (D-glucuronic acid or L-iduronicacid-N-acetyl-o-galactosamine), keratan sulfate(D-galactose-N-acetyl-D-glucosamine sulfate), and heparan sulfate(D-glucuronic acid or L-iduronic acid-N-acetyl-D-glucosamine).“Mucopolysaccharide” is a term that is synonymous withglycosaminoglycan.

By “glycoprotein” is meant a protein or polypeptide that is covalentlylinked to one or more carbohydrate molecules. Glycoproteins includeproteoglycans and many, if not most, of the important integral membraneproteins protruding through the exterior leaflet into the extracellularspace, as well as many, if not most, of the secreted proteins.

By “proteoglycan” is meant any of a diverse group of macromoleculescomprising proteins and glycosaminoglycans. “Mucoprotein” is a term thatis synonymous with proteoglycan.

“Isotope labeled substrate” includes any isotope-labeled precursormolecule that is able to be incorporated into a molecule of interest ina living system. Examples of isotope labeled substrates include, but arenot limited to, ²H₂O, ³H₂O, H₂ ¹⁸O, ²H-glucose, ²H-labeled amino acids,³H-labeled amino acids, ²H-labeled organic molecules, ³H-labeled organicmolecules, ¹³C-labeled organic molecules, ¹⁴C-labeled organic molecules,¹³CO₂, ¹⁴CO₂, ¹⁵N-labeled organic molecules and ¹⁵NH₃.

“Labeled sugar” refers to a sugar incorporating one or more stable orradioactive isotopes. As used herein, the term “labeled sugar” is usedinterchangeably with “isotopically labeled sugar.”

“Labeled fatty acid” refers to a fatty acid incorporating one or morestable or radioactive isotopes. As used herein, the term “labeled fattyacid” is used interchangeably with “isotopically labeled fatty acid.”

“Labeled Water” or “heavy water” includes water labeled with one or morespecific heavy isotopes of either hydrogen or oxygen. Specific examplesof labeled water include, but are not limited to, ²H₂O, ³H₂O, and H₂¹⁸O. As used herein, the term “labeled water” is used interchangeablywith “isotopically labeled water.”

“Deuterated water” refers to water incorporating one or more ²Hisotopes.

“Labeled glucose” refers to glucose labeled with one or more stable orradioactive isotopes. Specific examples of labeled glucose or ²H-labeledglucose include, but are not limited to, [6,6-²H₂]glucose,[1-²H₁]glucose, and [1,2,3,4,5,6-²H₇]glucose.

“Administer[ed]” includes a living system exposed to a compound,combination of compounds, or mixture of compounds. Such exposure can befrom, but is not limited to, topical application, oral ingestion,inhalation, subcutaneous injection, intraperitoneal injection,intravenous injection, and intraarterial injection, in animals or otherhigher organisms.

By “secondary therapeutic action” is meant a biological response to acompound, combination of compounds, or mixture of compounds that wasunanticipated or unexpected. Often, the biological response to acompound has been previously established or was explicitly predicted orcan be reasonably predicted from empirical data or other sources ofinformation, or was used as the basis to discover and/or develop thecompound. In the context of the present invention, a secondarytherapeutic action is one that was not explicitly established orpredicted or cannot be clearly predicted from pre-existing empiricaldata or other sources of information. Such a secondary therapeuticaction may constitute a “new use” or “new indication” of the compoundwhen another therapeutic action has been previously detected orestablished.

By “toxic effect” is meant an adverse response by a living system to acompound. In the context of the present invention, a toxic effect is onethat is unanticipated or unexpected in response to a compound and thatmight affect the therapeutic use and/or potential use of the compound.

An “individual” is a vertebrate, preferably a mammal, more preferably ahuman.

By “mammal” is meant any member of the class Mammalia including, withoutlimitation, humans and nonhuman primates such as chimpanzees and otherapes and monkey species; farm animals such as cattle, sheep, pigs, goatsand horses; domestic mammals such as dogs and cats; laboratory animalsincluding rodents such as mice, rats and guinea pigs, and the like. Theterm does not denote a particular age or sex. Thus, adult and newbornsubjects, as well as fetuses, whether male or female, are intended to becovered.

“At least partially identified” in the context of drug discovery anddevelopment means at least one clinically relevant pharmacologicalcharacteristic of a compound has been identified using one or more ofthe methods of the present invention. This characteristic may be adesirable one, for example, increasing or decreasing molecular fluxrates through a metabolic pathway that contributes to a disease process,altering signal transduction pathways or cell surface receptors thatalter the activity of metabolic pathways relevant to a disease,inhibiting activation of an enzyme and the like. Alternatively, apharmacological characteristic of a compound may be an undesirable onefor example, the production of one or more toxic effects. There are aplethora of desirable and undesirable characteristics of compounds wellknown to those skilled in the art and each will be viewed in the contextof the particular compound being developed and the targeted disease. Ofcourse, a compound can be more than at least partially identified, forexample, when several characteristics have been identified (desirable orundesirable or both) that are sufficient to support a particularmilestone decision point along the drug development pathway. Suchmilestones include, but are not limited to, pre-clinical decisions forin vitro to in vivo transition, pre-IND filing go/no go decision, phaseI to phase II transition, phase IIa to phase IIb transition, phase II tophase III transition, NDA filing, and FDA approval for marketing.Therefore, “at least partially” identified includes the identificationof one or more pharmacological characteristics useful in evaluating acompound in the drug discovery/drug development process. Apharmacologist or physician or other researcher may evaluate all or aportion of the identified desirable and undesirable characteristics of acompound to establish its therapeutic index. This may be accomplishedusing procedures well known in the art.

“Manufacturing compounds” in the context of the present inventionincludes any means, well known to those skilled in the art, employed forthe making of a compound as a product. A product is not limited to afinal approved therapeutic agent but may constitute a non-final compoundin the DDDA process, e.g., a prodrug or chemical intermediary (i.e., alead compound that has been out-licensed for further optimization),whose manufacture is in response to a commercial need. Manufacturingprocesses include, but are not limited to, medicinal chemical synthesis(i.e., synthetic organic chemistry), combinatorial chemistry,biotechnology methods such as hybridoma monoclonal antibody production,recombinant DNA technology, and other techniques well known to theskilled artisan, Such a product may be a final drug agent that ismarketed for therapeutic use, a component of a combination product thatis marketed for therapeutic use, or any intermediate product used in thedevelopment of the final drug agent product, whether as part of acombination product or a single product.

III. Methods of the Invention

The present invention is directed to methods of detecting actions,particularly unintended or unexpected actions, of compounds in livingsystems by measuring the molecular flux rates of one or more moleculesin one or more metabolic pathways of interest within a living system.First, at least one isotope-labeled substrate molecule is administeredto one or more cells, tissues or organisms for a period of timesufficient to be incorporated in vivo into one or more targetedmolecules of interest within one or more targeted metabolic pathways. Inone embodiment, the isotope-labeled substrate molecules are labeled withone or more stable isotopes (i.e., non-radioactive isotope). In anotherembodiment, the isotope-labeled substrate molecule is labeled with oneor more radioactive isotopes. In yet another embodiment, both stable andradioactive isotopes are used to label one or more isotope-labeledsubstrate molecules.

The targeted molecule of interest is obtained by biochemical isolationprocedures from the one or more cells, tissues, or organisms, and isidentified by mass spectrometry, liquid scintillation, or by other meansknown in the art. The relative and absolute abundances of the ionswithin the mass isotopomeric envelope corresponding to each identifiedmolecule of interest (i.e., the isotopic content and/or pattern of themolecule or the rate of change of the isotopic content and/or pattern ofthe molecule) are quantified. In one embodiment, the relative andabsolute abundances of the ions within the mass isotopomeric envelopecorresponding to each identified molecule of interest are quantified bymass spectrometry. Molecular flux rates through the targeted metabolicpathways of interest are then calculated by use of equations known inthe art and discussed, infra. Molecular flux rates through the targetedmetabolic pathways of interest are compared in the presence or absenceof exposure to one or more compounds, combinations of compounds, ormixtures of compounds, or in response to different levels of exposure tocompounds, or in response to different levels of exposure tocombinations of compounds or mixtures of compounds.

A. ADMINISTERING ISOTOPE-LABELED PRECURSOR(S)

As a first step in the methods of the invention, isotope-labeledprecursors are administered.

1. Administering an Isotope-Labeled Precursor Molecule

Modes of administering the one or more isotope-labeled substrates mayvary, depending upon the absorptive properties of the isotope-labeledsubstrate and the specific biosynthetic pool into which each compound istargeted. Precursors may be administered to organisms, plants andanimals including humans directly for in vivo analysis. In addition,precursors may be administered in vitro to living cells or ex vivo intissues or organs. Specific types of living cells include hepatocytes,adipocytes, myocytes, fibroblasts, neurons, pancreatic β-cells,intestinal epithelial cells, leukocytes, lymphocytes, erythrocytes,microbial cells and any other cell-type that can be maintained alive andfunctional in vitro.

Generally, an appropriate mode of administration is one that produces asteady state level of precursor within the biosynthetic pool and/or in areservoir supplying such a pool for at least a transient period of time.Intravascular or oral routes of administration are commonly used toadminister such precursors to organisms, including humans. Other routesof administration, such as subcutaneous or intra-muscularadministration, optionally when used in conjunction with slow releaseprecursor compositions, formulations, or techniques, are alsoappropriate. Compositions for injection are generally prepared insterile pharmaceutical excipients.

a. Labeled Precursor Molecules

(1) Isotope Labels

The first step in measuring molecular flux rates involves administeringone or more isotope-labeled precursor molecules to one or more cells,tissues, or organisms. The isotope labeled precursor molecule maycontain a stable isotope or a radioisotope. Isotope labels that can beused in accordance with the methods of the present invention include,but are not limited to, ²H, ¹³C, ¹⁵N, ¹⁸O, ³H, ¹⁴C, ³⁵S, ³²P, ¹²⁵I,¹³¹I, or other isotopes of elements present in organic systems. Theseisotopes, and others, are suitable for all classes of chemicals (i.e.,precursor molecules) envisioned for use in the present invention. Suchprecursor molecules include, but are not limited to, amino acidprecursors, protein precursors, lipid precursors, carbohydrateprecursors, nucleic acid precursors, porphyrin precursors,glycosaminoglycan precursors, and proteoglycan precursors (see examplesof each, infra).

In one embodiment, the isotope label is ²H.

(2) Precursor Molecules (Isotope-Labeled Substrates)

The precursor molecule may be any molecule having an isotope label thatis incorporated into a molecule of interest by passage through ametabolic pathway in vivo in a living system. Precursor molecules thatmay be used include, without limitation: H₂O; CO₂; NH₃; acetyl CoA (toform cholesterol, fatty acids); ribonucleic acids (to form RNA);deoxyribonucleic acids (to form DNA); glucose (to form glycogen); aminoacids (to form peptides/proteins); phosphoenol-pyruvate (to formglucose/UDP-glucose); and glycine/succinate (to form porphyrinderivatives). Isotope labels may be used to modify all precursormolecules disclosed herein to form isotope-labeled precursor molecules.

The entire precursor molecule may be incorporated into one or moremolecules of interest within a metabolic pathway. Alternatively, aportion of the precursor molecule may be incorporated into one or moremolecules of interest.

The individual being administered one or more isotope labeled substrates(i.e., one or more precursor molecules) may be a mammal. In onevariation, the mammal may be a rodent (rat or mouse), primate, hamster,guinea pig, dog, or pig. The mammal may be wild-type. In anotherembodiment, the mammal may be an engineered animal including, but notlimited to, a transgenic animal, a gene knock-out animal, or a geneknock-in animal. In one embodiment, the mammal may be healthy. Inanother embodiment, the mammal may have a disease or medical condition.Mammals having a disease or having a medical condition may have acongenital disease or medical condition or an acquired disease ormedical condition. Examples of mammals having either a congenitaldisease or medical condition or an acquired disease or medical conditionare well known to those of skill in the art.

In still another embodiment, the mammal may be a human.

i. Protein Precursors

A protein precursor molecule may be any protein precursor molecule knownin the art. These precursor molecules may be amino acids, CO₂, NH₃,glucose, lactate, H₂O, acetate, and fatty acids.

The isotope label may include specific heavy isotopes of elementspresent in biomolecules, such as ²H, ¹³C, ¹⁵N, ¹⁸O, ³³S, ³⁴S, or maycontain other isotopes of elements present in biomolecules such as ³H,¹⁴C, ³⁵S, ¹²⁵I, or ¹³¹I.

Precursor molecules of proteins may include one or more amino acids. Theprecursor may be any amino acid. The precursor molecule may be a singlyor multiply deuterated amino acid. The precursor molecule may be one ormore of ¹³C-lysine, ¹⁵N-histidine, ¹³C-serine, ¹³C-glycine, ²H-leucine,¹⁵N-glycine, ¹³C-leucine, ²H₅-histidine, and any deuterated amino acid.By way of example, isotope labeled protein precursors include, but arenot limited to ²H₂O, H₂ ¹⁸O, ¹⁵NH₃, ¹³CO₂, H¹³CO₃, ²H-labeled aminoacids, ¹³C labeled amino acids, ¹⁵N labeled amino acids, ¹⁸O labeledamino acids, ³³S or ³⁴S labeled amino acids, ³H₂O, ³H-labeled aminoacids, and ¹⁴C labeled amino acids. Labeled amino acids may beadministered, for example, undiluted or diluted with non-labeled aminoacids. All isotope labeled precursors may be purchased commercially, forexample, from Cambridge Isotope Labs (Andover, Mass.).

Protein precursor molecules may also include any precursor forpost-translational or pre-transiationally modified amino acids. Theseprecursors include but are not limited to precursors of methylation suchas glycine, serine or H₂O; precursors of hydroxylation, such as H₂O orO₂; precursors of phosphorylation, such as phosphate, H₂O or O₂;precursors of prenylation, such as fatty acids, acetate, H₂O, ethanol,ketone bodies, glucose, or fructose; precursors of carboxylation, suchas CO₂, O₂, H₂O, or glucose; precursors of acetylation, such as acetate,ethanol, glucose, fructose, lactate, alanine, H₂O, CO₂, or O₂; and otherpre or post-translational modifications known in the art.

The degree of labeling present in free amino acids may be determinedexperimentally, or may be assumed based on the number of labeling sitesin an amino acid. For example, when using hydrogen isotopes as a label,the labeling present in C—H bonds of free amino acid or, morespecifically, in tRNA-amino acids, during exposure to ²H₂O in body watermay be identified. The total number of C—H bonds in each non essentialamino acid is known—e.g., 4 in alanine, 2 in glycine, etc.

The precursor molecule for proteins may be water. The hydrogen atoms onC—H bonds are the hydrogen atoms on amino acids that are useful formeasuring protein synthesis from ²H₂O since the O—H and N—H bonds ofproteins are labile in aqueous solution. As such, the exchange of²H-label from ²H₂O into O—H or N—H bonds occurs without the synthesis ofproteins from free amino acids as described above. C—H bonds undergoincorporation from H₂O into free amino acids during specificenzyme-catalyzed intermediary metabolic reactions. The presence of²H-label in C—H bonds of protein-bound amino acids after ²H₂Oadministration therefore means that the protein was assembled from aminoacids that were in the free form during the period of ²H₂Oexposure—i.e., that the protein is newly synthesized. Analytically, theamino acid derivative used must contain all the C—H bonds but mustremove all potentially contaminating N—H and O—H bonds.

Hydrogen atoms from body water may be incorporated into free aminoacids. ²H or ³H from labeled water (i.e., ²H₂O or ³H₂O) can enter intofree amino acids in the cell through the reactions of intermediarymetabolism, but ²H or ³H cannot enter into amino acids that are presentin peptide bonds or that are bound to transfer RNA. Free essential aminoacids may incorporate a single hydrogen atom from body water into theα-carbon C—H bond, through rapidly reversible transamination reactions.Free non-essential amino acids contain a larger number of metabolicallyexchangeable C—H bonds, of course, and are therefore expected to exhibithigher isotopic enrichment values per molecule from ²H₂O or ³H₂O innewly synthesized proteins.

One of skill in the art will recognize that labeled hydrogen atoms frombody water may be incorporated into other amino acids via otherbiochemical pathways. For example, it is known in the art that hydrogenatoms from water may be incorporated into glutamate via synthesis of theprecursor α-ketogiutarate in the citric acid cycle. Glutamate, in turn,is known to be the biochemical precursor for glutamine, proline, andarginine. By way of another example, hydrogen atoms from body water maybe incorporated into post-translationally modified amino acids, such asthe methyl group in 3-methyl-histidine, the hydroxyl group inhydroxyproline or hydroxylysine, and others. Other amino acid synthesispathways are known to those of skill in the art.

Oxygen atoms (H₂ ¹⁸O) may also be incorporated into amino acids throughenzyme-catalyzed reactions. For example, oxygen exchange into thecarboxylic acid moiety of amino acids may occur during enzyme catalyzedreactions. Incorporation of labeled oxygen into amino acids is known toone of skill in the art. Oxygen atoms may also be incorporated intoamino acids from ¹⁸O₂ through enzyme catalyzed reactions (includinghydroxyproline, hydroxylysine or other post-translationally modifiedamino acids).

Hydrogen and oxygen labels from labeled water may also be incorporatedinto amino acids through post-translational modifications. In oneembodiment, the post-translational modification may already includelabeled hydrogen or oxygen through biosynthetic pathways prior topost-translational modification. In another embodiment, thepost-translational modification may incorporate labeled hydrogen,oxygen, carbon, or nitrogen from metabolic derivatives involved in thefree exchange labeled hydrogens from body water, either before or aftera post-translational modification step (e.g., methylation,hydroxylation, phosphorylation, prenylation, sulfation, carboxylation,acetylation or other known post-translational modifications).

Protein precursors that are suitable for administration into a subjectinclude, but are not limited to H₂O, CO₂, NH₃ and HCO₃, in addition tothe standard amino acids found in proteins.

The individual being administered protein precursors may be a mammal. Inone variation, the mammal may be a rodent (rat or mouse), primate,hamster, guinea pig, dog, or pig. The mammal may be wild-type. Inanother embodiment, the mammal may be an engineered animal including,but not limited to, a transgenic animal, a gene knock-out animal, or agene knock-in animal. In one embodiment, the mammal may be healthy. Inanother embodiment, the mammal may have a disease or medical condition.Mammals having a disease or having a medical condition may have acongenital disease or medical condition or an acquired disease ormedical condition. Examples of mammals having either a congenitaldisease or medical condition or an acquired disease or medical conditionare well known to those of skill in the art.

In still another embodiment, the mammal may be a human.

ii. Precursors of Organic Metabolites

Precursors of organic metabolites may be any precursor molecule capableof entering into the organic metabolite pathway. Organic metabolites andorganic metabolite precursors include, but are not limited to, H₂O, CO₂,NH₃, HCO₃, amino acids, monosaccharides, carbohydrates, lipids, fattyacids, nucleic acids, glycolytic intermediates, acetic acid, andtricarboxylic acid cycle intermediates.

Isotope labeled organic metabolite precursors include, but are notlimited to, ²H₂O, ¹⁵NH₃, ¹³CO₂, H¹³CO₃, ²H-labeled amino acids,¹³C-labeled amino acids, ¹⁵N-labeled amino acids, H₂ ¹⁸O, ¹⁸O-labeledamino acids, ³³S or ³⁴S-labeled amino acids, ³H₂O, ³H-labeled aminoacids, ¹⁴C-labeled amino acids, ¹⁴CO₂, and H¹⁴CO₂.

Organic metabolite precursors may also be administered directly. Massisotopes that may be useful in mass isotope labeling of organicmetabolite precursors include, but are not limited to, ²H, ³H, ¹³C, ¹⁴C,¹⁵N, ¹⁸O, ³³S, ³⁴S, ³⁵S, ³²P, ¹²⁵I, ¹³¹I, or other isotopes of elementspresent in organic systems. It is often desirable, in order to avoidmetabolic loss of isotope labels, that the isotope-labeled atom(s) berelatively non-labile or at least behave in a predictable manner withinthe subject. By administering the isotope-labeled precursors to thebiosynthetic pool, the isotope-labeled precursors can become directlyincorporated into organic metabolites formed in the pool.

The individual being administered organic metabolite precursors may be amammal. In one variation, the mammal may be a rodent (rat or mouse),primate, hamster, guinea pig, dog, or pig. The mammal may be wild-type.In another embodiment, the mammal may be an engineered animal including,but not limited to, a transgenic animal, a gene knock-out animal, or agene knock-in animal. In one embodiment, the mammal may be healthy. Inanother embodiment, the mammal may have a disease or medical condition.Mammals having a disease or having a medical condition may have acongenital disease or medical condition or an acquired disease ormedical condition. Examples of mammals having either a congenitaldisease or medical condition or an acquired disease or medical conditionare well known to those of skill in the art.

In still another embodiment, the mammal may be a human.

iii. Precursors of Nucleic Acids

Precursors of nucleic acids (i.e., RNA, DNA) are any substrates suitablefor incorporation into RNA and/or DNA synthetic pathways. Examples ofsubstrates useful in labeling the deoxyribose ring of DNA include, butare not limited to, [6,6-²H₂]glucose, [U-¹³C₆] glucose and [2-¹³C₁]glycerol (see U.S. Pat. No. 6,461,806, herein incorporated byreference). Labeling of the deoxyribose is superior to labeling of theinformation-carrying nitrogen bases in DNA because it avoids variabledilution sources. The stable isotope labels are readily detectable bymass spectrometric techniques.

In one embodiment, a stable isotope label is used to label thedeoxyribose ring of DNA from glucose, precursors of glucose-6-phosphateor precursors of ribose-5-phosphate. In embodiments where glucose isused as the starting material, suitable labels include, but are notlimited to, deuterium-labeled glucose such as [6,6-²H₂] glucose, [1-²H₁]glucose, [3-²H₁] glucose, [²H₇] glucose, and the like; ¹³C-1 labeledglucose such as [1-¹³C₁] glucose, [U-¹³C₆] glucose and the like; and¹⁸O-labeled glucose such as [1-¹⁸O₂] glucose and the like.

In embodiments where a glucose-5-phosphate precursor or aribose-5-phosphate precursor is desired, a gluconeogenic precursor or ametabolite capable of being converted to glucose-5-phosphate orribose-5-phosphate can be used. Gluconeogenic precursors include, butare not limited to, ¹³C-labeled glycerol such as [2-¹³C₁] glycerol andthe like, a ¹³C-labeled amino acid, deuterated water (²H₂O) and¹³C-labeled lactate, alanine, pyruvate, propionate or other non-aminoacid precursors for gluconeogenesis. Metabolites which are converted toglucose-5-phosphate or ribose-5-phosphate include, but are not limitedto, labeled (²H or ¹³C) hexoses such as [1-²H₁] galactose, [U-¹³C]fructose and the like; labeled (²H or ¹³C) pentoses such as [1-¹³C₁]ribose, [1-²H₁] xylitol and the like, labeled (²H or ¹³C) pentosephosphate pathway metabolites such as [1-²H₁] seduheptalose and thelike, and labeled (²H or ¹³C) amino sugars such as [U-¹³C] glucosamine,[1-²H₁] N-acetyl-glucosamine and the like.

The present invention also encompasses stable isotope labels which labelpurine and pyrimidine bases of DNA through the de novo nucleotidesynthesis pathway. Various building blocks for endogenous purinesynthesis can be used to label purines and they include, but are notlimited to, ¹⁵N-labeled amino acids such as [¹⁵N] glycine, [¹⁵N]glutamine, [¹⁵N] aspartate and the like, ¹³C-labeled precursors such as[1-¹³C₁] glycone, [3-¹³C₁]acetate, [¹³C]HCO₃, [¹³C] methionine and thelike, and H-labeled precursors such as ²H₂O. Various building blocks forendogenous pyrimidine synthesis can be used to label pyrimidines andthey include, but are not limited to, ¹⁵N-labeled amino acids such as[¹⁵N] glutamine and the like, ¹³C-labeled precursors such as [¹³C]HCO₃,[U-¹³C₄] aspartate and the like, and ²H-labeled precursors (e.g., ²H₂O).

It is understood by those skilled in the art that in addition to thelist above, other stable isotope labels which are substrates orprecursors for any pathways which result in endogenous labeling of DNAare also encompassed within the scope of the invention. The labelssuitable for use in the present invention are generally commerciallyavailable or can be synthesized by methods well known in the art.

The individual being administered nucleic acid precursors may be amammal. In one variation, the mammal may be a rodent (rat or mouse),primate, hamster, guinea pig, dog, or pig. The mammal may be wild-type.In another embodiment, the mammal may be an engineered animal including,but not limited to, a transgenic animal, a gene knock-out animal, or agene knock-in animal. In one embodiment, the mammal may be healthy. Inanother embodiment, the mammal may have a disease or medical condition.Mammals having a disease or having a medical condition may have acongenital disease or medical condition or an acquired disease ormedical condition. Examples of mammals having either a congenitaldisease or medical condition or an acquired disease or medical conditionare well known to those of skill in the art. In still anotherembodiment, the mammal may be a human.

iv. Water as a Precursor Molecule

Water is a precursor of proteins and many organic metabolites. As such,labeled water may serve as a precursor in the methods taught herein.

H₂O availability is probably never limiting for biosynthetic reactionsin a cell (because H₂O represents close to 70% of the content of cells,or a 35 Molar concentration), but hydrogen and oxygen atoms from H₂Ocontribute stoichiometrically to many reactions involved in biosyntheticpathways: e.g.: R—CO—CH₂—COOH+NADPH+H₂O→R—CH₂CH₂COOH (fatty acidsynthesis).

As a consequence, isotope labels provided in the form of H- orO-isotope-labeled water is incorporated into biological molecules aspart of synthetic pathways. Hydrogen incorporation can occur in twoways: into labile positions in a molecule (i.e., rapidly exchangeable,not requiring enzyme-catalyzed reactions) or into stable positions(i.e., not rapidly exchangeable, requiring enzyme catalysis). Oxygenincorporation occurs in stable positions.

Some of the hydrogen-incorporating steps from cellular water into C—Hbonds in biological molecules only occur during well-definedenzyme-catalyzed steps in the biosynthetic reaction sequence, and arenot labile (i.e., exchangeable with solvent water in the tissue) oncepresent in the mature end-product molecules. For example, the C—H bondson glucose are not exchangeable in solution. In contrast, each of thefollowing C—H positions exchanges with body water during reversal ofspecific enzymatic reactions: C-1 and C-6, in the oxaloacetate/succinatesequence in the Krebs' cycle and in the lactate/pyruvate reaction; C-2,in the glucose-6-phosphate/fructose-6-phosphate reaction; C-3 and C-4,in the glyceraldehyde-3-phosphate/dihydroxyacetone-phosphate reaction;C-5, in the 3-phosphoglycerate/glyceraldehyde-3-phosphate andglucose-6-phosphate/fructose-6-phosphate reactions.

Labeled hydrogen or oxygen atoms from water that are covalentlyincorporated into specific non-labile positions of a molecule therebyreveals the molecule's “biosynthetic history”—i.e., label incorporationsignifies that the molecule was synthesized during the period thatisotope-labeled water was present in cellular water.

Conversely, labile hydrogens (non-covalently associated or present inexchangeable covalent bonds) in these biological molecules do not revealthe molecule's biosynthetic history. Labile hydrogen atoms can be easilyremoved by incubation with unlabelled water (H₂O) (i.e., by reversal ofthe same non-enzymatic exchange reactions through which ²H or ³H wasincorporated in the first place), as the following reactiondemonstrates:

As a consequence, a potentially contaminating hydrogen label that doesnot reflect biosynthetic history, but is incorporated via non-syntheticexchange reactions, can be easily removed in practice by incubation withnatural abundance H₂O.

Analytic methods are available for measuring quantitatively theincorporation of labeled hydrogen atoms into biological molecules (e.g.,liquid scintillation counting for ³H; mass spectrometry or NMRspectroscopy for ²H and ¹⁸O). For further discussions on the theory ofisotope-labeled water incorporation, see, for example, Jungas R L.Biochemistry. 1968 7:3708-17, incorporated herein by reference.

Labeled water may be readily obtained commercially. For example, ²H₂Omay be purchased from Cambridge Isotope Labs (Andover, Mass.), and ³H₂Omay be purchased, e.g., from New England Nuclear, Inc. In general, ²H₂Ois non-radioactive and thus, presents fewer toxicity concerns thanradioactive ³H₂O. ²H₂O may be administered, for example, as a percent oftotal body water, e.g., 1% of total body water consumed (e.g., for 3liters water consumed per day, 30 microliters ²H₂O is consumed). If ³H₂Ois utilized, then a non-toxic amount, which is readily determined bythose of skill in the art, is administered.

Relatively high body water enrichments of ²H₂O (e.g., 1-10% of the totalbody water is labeled) may be achieved relatively inexpensively usingthe techniques of the invention. This water enrichment is relativelyconstant and stable as these levels are maintained for weeks or monthsin humans and in experimental animals without any evidence of toxicity.This finding in a large number of human subjects (>100 people) iscontrary to previous concerns about vestibular toxicities at high dosesof ²H₂O. The Applicant has discovered that as long as rapid changes inbody water enrichment are prevented (e.g., by initial administration insmall, divided doses), high body water enrichments of ²H₂O can bemaintained with no toxicities. For example, the low cost of commerciallyavailable ²H₂O allows long-term maintenance of enrichments in the 1-5%range at relatively low expense (e.g., calculations reveal a lower costfor 2 months labeling at 2% ²H₂O enrichment, and thus 7-8% enrichment inthe alanine precursor pool, than for 12 hours labeling of ²H-leucine at10% free leucine enrichment, and thus 7-8% enrichment in leucineprecursor pool for that period).

Relatively high and relatively constant body water enrichments foradministration of H₂ ¹⁸O may also be accomplished, since the ¹⁸O isotopeis not toxic, and does not present a significant health risk as aresult.

Isotope-labeled water may be administered via continuous isotope-labeledwater administration, discontinuous isotope-labeled wateradministration, or after single or multiple administration ofisotope-labeled water administration. In continuous isotope-labeledwater administration, isotope-labeled water is administered to anindividual for a period of time sufficient to maintain relativelyconstant water enrichments over time in the individual. For continuousmethods, labeled water is optimally administered for a period ofsufficient duration to achieve a steady state concentration (e.g., 3-8weeks in humans, 1-2 weeks in rodents).

In discontinuous isotope-labeled water administration, an amount ofisotope-labeled water is measured and then administered, one or moretimes, and then the exposure to isotope-labeled water is discontinuedand wash-out of isotope-labeled water from body water pool is allowed tooccur. The time course of delabeling may then be monitored. Water isoptimally administered for a period of sufficient duration to achievedetectable levels in biological molecules.

Isotope-labeled water may be administered to an individual or tissue invarious ways that are well known in the art. For example,isotope-labeled water may be administered orally, parenterally,subcutaneously, intravascularly (e.g., intravenously, intraarterially),or intraperitoneally. Several commercial sources of ²H₂O and H₂ ¹⁸O areavailable, including Isotec, Inc. (Miamisburg Ohio, and CambridgeIsotopes, Inc. (Andover, Mass.). The isotopic content of isotope labeledwater that is administered can range from about 0.001% to about 20% anddepends upon the analytic sensitivity of the instrument used to measurethe isotopic content of the biological molecules. For oraladministration, 4% ²H₂O in drinking water is administered. For humanadministration, 50 mL H₂O² is administered.

The individual being administered labeled water may be a mammal. In onevariation, the mammal may be a rodent (rat or mouse), primate, hamster,guinea pig, dog, or pig. The mammal may be wild-type. In anotherembodiment, the mammal may be an engineered animal including, but notlimited to, a transgenic animal, a gene knock-out animal, or a geneknock-in animal. In one embodiment, the mammal may be healthy. Inanother embodiment, the mammal may have a disease or medical condition.Mammals having a disease or having a medical condition may have acongenital disease or medical condition or an acquired disease ormedical condition. Examples of mammals having either a congenitaldisease or medical condition or an acquired disease or medical conditionare well known to those of skill in the art.

In still another embodiment, the mammal may be a human.

v. Precursors of Carbohydrates

Compositions comprising carbohydrates may include monosaccharides,polysaccharides, or other compounds attached to monosaccharides orpolysaccharides.

Isotope labels may be incorporated into carbohydrates or carbohydratederivatives by biochemical pathways known in the art. These includemonosaccharides (including, but not limited to, glucose and galactose),amino sugars (such as N-Acetyl-Galactosamine), polysaccharides (such asglycogen), glycoproteins (such as sialic acid) glycolipids (such asgalactocerebrosides), and glycosaminoglycans (such as hyaluronic acid,chondroitin-sulfate, and heparan-sulfate).

²H-labeled sugars may be administered to an individual asmonosaccharides or as polymers comprising monosaccharide residues.Labeled monosaccharides may be readily obtained commercially (e.g.,Cambridge Isotopes, Mass.).

Relatively low quantities of compounds comprising ²H-labeled sugars needbe administered. Quantities may be on the order of milligrams, 10¹ mg,10² mg, 10³ mg, 10⁴ mg, 10⁵ mg, or 10⁶ mg. ²H-labeled sugar enrichmentmay be maintained for weeks or months in humans and in animals withoutany evidence of toxicity. The low cost of commercially available labeledmonosaccharides, and low quantity that need to be administered, allowmaintenance of enrichments at low expense.

In one embodiment, the labeled sugar is glucose. Glucose is metabolizedby glycolysis and the citric acid cycle. Glycolysis releases most of theH-atoms from the C—H bonds of glucose; oxidation via the citric acidcycle ensures that all H-atoms are released to H₂O. The loss of ³H- or²H-label by glucose has been used to assess glycolysis, an intracellularmetabolic pathway for glucose. Some investigators have used release of³H from intravenously administered ³H-glucose into ³H₂O as a measure ofglycolysis. Release of ²H-glucose into ²H₂O has not been describedpreviously, ostensibly because of the expectation that the body waterpool is too large relative to ²H administration in labeled glucose toachieve measurable ²H₂O levels. The Applicant has discovered otherwise,demonstrating that the release of ²H-glucose into ²H₂O can be measured(see U.S. patent application Ser. No. 10/701,990, herein incorporated byreference). In a further variation, the labeled glucose may be[6,6-²H₂]glucose, [1-²H₁]glucose, and [1,2,3,4,5,6-²H₇]glucose.

In another variation, labeled sugar comprises fructose or galactose.Fructose enters glycolysis via the fructose 1-phosphate pathway, andsecondarily through phosphorylation to fructose 6-phosphate byhexokinase. Galactose enters glycolysis via the galactose to glucoseinterconversion pathway.

Any other sugar is envisioned by the present invention. Contemplatedmonosaccharides, include, but are not limited to, trioses, pentoses,hexose, and higher order monosaccharides. Monosaccharides furtherinclude, but are not limited to, aldoses and ketoses.

In another variation, compounds comprising polysaccharides may beadministered. The polymers may comprise polysaccharides. For example,labeled glycogen, a polysaccharide, comprises glucose residues. Inanother variation, labeled polysaccharides may be introduced. As furthervariation, labeled sugar monomers may be administered as a component ofsucrose (glucose α-(1, 2)-fructose), lactose (galactose β-(1,4)-glucose), maltose (glucose α-(1, 4)-glucose), starch (glucosepolymer), or other polymers.

In another variation, the labeled sugar may be administered orally, bygavage, intraperitoneally, intravascularly (e.g., intravenously,intraarterially), subcutaneously, or other bodily routes. In particular,the sugars may be administered to an individual orally, optionally aspart of a food or drink.

The individual being administered carbohydrate precursors may be amammal. In one variation, the mammal may be a rodent (rat or mouse),primate, hamster, guinea pig, dog, or pig. The mammal may be wild-type.In another embodiment, the mammal may be an engineered animal including,but not limited to, a transgenic animal, a gene knock-out animal, or agene knock-in animal. In one embodiment, the mammal may be healthy. Inanother embodiment, the mammal may have a disease or medical condition.Mammals having a disease or having a medical condition may have acongenital disease or medical condition or an acquired disease ormedical condition. Examples of mammals having either a congenitaldisease or medical condition or an acquired disease or medical conditionare well known to those of skill in the art.

In still another embodiment, the mammal may be a human.

vi. Precursors of Lipids and Other Fats

Labeled precursors of lipids may include any precursor in lipidbiosynthesis. The precursor molecules of lipids may be CO₂, NH₃,glucose, lactate, H₂O, acetate, and fatty acids. The precursor may alsoinclude labeled water, e.g., ²H₂O, which is a precursor for fatty acids,glycerol moiety of acyl-glycerols, cholesterol and its derivatives; ¹³Cor ²H-labeled fatty acids, which are precursors for triglycerides,phospholipids, cholesterol ester, coamides and other lipids; ¹³C- or²H-acetate, which is a precursor for fatty acids and cholesterol; ¹⁸O₂(e.g., g from H₂ ¹⁸O), which is a precursor for fatty acids,cholesterol, acyl-glycerides, and certain oxidatively modified fattyacids (such as peroxides) by either enzymatically catalyzed reactions orby non-enzymatic oxidative damage (e.g., to fatty acids); ¹³C- or²H-glycerol, which is a precursor for acyl-glycerides; ¹³C- or²H-labeled acetate, ethanol, ketone bodies or fatty acids, which areprecursors for endogenously synthesized fatty acids, cholesterol andacylglycerides; and ²H or ¹³C-labeled cholesterol or its derivatives(including bile acids and steroid hormones). All isotope labeledprecursors may be purchased commercially, for example, from CambridgeIsotope Labs (Andover, Mass.).

Complex lipids, such as glycolipids and cerebrosides, can also belabeled from precursors, including ²H₂O, which is a precursor for thesugar-moiety of cerebrosides (including, but not limited to,N-acetylgalactosamine, N-acetylglucosamine-sulfate, glucuronic acid, andglucuronic acid-sulfate), the fatty acyl-moiety of cerebrosides and thesphingosine moiety of cerebrosides; ²H- or ¹³C-labeled fatty acids,which are precursors for the fatty acyl moiety of cerebrosides,glycolipids and other derivatives.

The precursor molecule may be or include components of lipids.

In one embodiment, ²H-labeled fatty acids may be administered to anindividual as fats or other substrates containing the labeled fattyacids. ²H-labeled fatty acids may be readily obtained commercially.Relatively low quantities of labeled fatty acids need be administered.Quantities may be on the order of milligrams, 10¹ mg, 10² mg, 10³ mg,10⁴ mg, 10⁵ mg, or 10⁶ mg. Fatty acid enrichment, particularly with ²H,may be maintained for weeks or months in humans and in animals withoutany evidence of toxicity. The low cost of commercially available labeledfatty acids, and low quantity that need to be administered, allowmaintenance of enrichments at low expense.

The release of labeled fatty acids, particularly ²H-fatty acid, tolabeled water, particularly ²H₂O, accurately reflects fat oxidation.Administration of modest amounts of labeled-fatty acid is sufficient tomeasure release of labeled hydrogen or oxygen to water. In particular,administration of modest amounts of ²H-fatty acid is sufficient tomeasure release of ²H to deuterated water.

In another variation, the labeled fatty acids may be administeredorally, by gavage, intraperitoneally, intravascularly (e.g.,intravenously, intraarterially), subcutaneously, or other bodily routes.In particular, the labeled fatty acids may be administered to anindividual orally, optionally as part of a food or drink.

The individual being administered lipid precursors may be a mammal. Inone variation, the mammal may be a rodent (rat or mouse), primate,hamster, guinea pig, dog, or pig. The mammal may be wild-type. Inanother embodiment, the mammal may be an engineered animal including,but not limited to, a transgenic animal, a gene knock-out animal, or agene knock-in animal. In one embodiment, the mammal may be healthy. Inanother embodiment, the mammal may have a disease or medical condition.Mammals having a disease or having a medical condition may have acongenital disease or medical condition or an acquired disease ormedical condition. Examples of mammals having either a congenitaldisease or medical condition or an acquired disease or medical conditionare well known to those of skill in the art.

In still another embodiment, the mammal may be a human.

vii. Precursors of Glycosaminoglycans and Proteoglycans

Glycosaminoglycans and proteoglycans are a complex class of biomoleculesthat play important roles in the extracellular space (e.g., cartilage,ground substance, and synovial joint fluid). Molecules in these classesinclude, for example, the large polymers built from glycosaminoglycandisaccharides, such as hyaluronan, which is a polymer composed of up to50,000 repeating units of hyaluronic acid (HA) disaccharide, a dimerthat contains N-acetyl-glucosamine linked to glucuronic acid;chondroitin-sulfate (CS) polymers, which are built from repeating unitsof CS disaccharide, a dimer that contains N-acetyl-galactosamine-sulfatelinked to glucuronic acid, heparan-sulfate polymers, which are builtfrom repeating units of heparan-sulfate, a dimer of N-acetyl (orN-sulfo)-glucosamine-sulfate linked to glucuronic acid; andkeratan-sulfate polymers, which are built from repeating units ofkeratan-sulfate disaccharide, a dimer that contains Nacetylglucosamine-sulfate liked to galactose. Proteoglycans containadditional proteins that are bound to a central hyaluronan polymer andother glycosaminoglycans, such as CS, that branch off of the centralhyaluronan chain.

Labeled precursors of glycosaminoglycans and proteoglycans include, butare not limited to, ²H₂O (incorporated into the sugar moieties,including N-acetylglucosamine, N-acetylgalactosamine, glucuronic acid,the various sulfates of N-acetylglucosamine and N-acetylgalactosamine,galactose, iduronic acid, and others), ¹³C- or ²H-glucose (incorporatedinto said sugar moieties), ²H- or ¹³C-fructose (incorporated into saidsugar moieties), ²H- or ¹³C-galactose (incorporated into said sugarmoieties), ¹⁵N-glycine, other ¹⁵N-labeled amino acids, or ¹⁵N-urea(incorporated into the nitrogen-moiety of said amino sugars, such asIV-acetylglycosamine, N-acetyl-galactosamine, etc.); ¹³C- or ²H-fattyacids, ¹³C- or ²H-ketone bodies, ¹³C-glucose, ¹³C-fructose, ¹⁸O₂ (e.g.,administered as H₂ ¹⁸O), ¹³C- or ²H-acetate (incorporated into theacetyl moiety of N-acetyl-sugars, such as N-acetyl-glucosamine orN-acetyl-galactosamine), and ¹⁸O or ³⁵S-labeled sulfate (incorporatedinto the sulfate moiety of chondroitin-sulfate, heparan-sulfate,keratan-sulfate, and other sulfate moieties). All isotope labeledprecursors may be purchased commercially, for example, from CambridgeIsotope Labs (Andover, Mass.),

The labeled glycosaminoglycan or proteoglycan precursors may beadministered orally, by gavage, intraperitoneally, intravascularly(e.g., intravenously, intraarterially), subcutaneously, or other bodilyroutes. In particular, the labeled glycosaminoglycan or proteoglycanprecursors may be administered to an individual orally, optionally aspart of a food or drink.

The individual being administered glycosaminoglycan or proteoglycanprecursors may be a mammal. In one variation, the mammal may be a rodent(rat or mouse), primate, hamster, guinea pig, dog, or pig. The mammalmay be wild-type. In another embodiment, the mammal may be an engineeredanimal including, but not limited to, a transgenic animal, a geneknock-out animal, or a gene knock-in animal. In one embodiment, themammal may be healthy. In another embodiment, the mammal may have adisease or medical condition. Mammals having a disease or having amedical condition may have a congenital disease or medical condition oran acquired disease or medical condition. Examples of mammals havingeither a congenital disease or medical condition or an acquired diseaseor medical condition are well known to those of skill in the art.

In still another embodiment, the mammal may be a human.

B. OBTAINING ONE OR MORE TARGETED MOLECULES OF INTEREST

In practicing the methods of the invention, in one aspect, targetedmolecules of interest are obtained from one or more cells, tissues, ororganisms according to methods known in the art. The methods may bespecific to a particular molecule of interest. Molecules of interest maybe isolated from one or more biological samples.

A plurality of molecules of interest may be acquired from the one ormore cells, tissues, or organisms. The one or more biological samplesmay be obtained, for example, by blood draw, urine collection, biopsy,or other methods known in the art. The one or more biological samplesmay be one or more biological fluids. The one or more molecules ofinterest also may be obtained from specific organs or tissues, such asmuscle, liver, adrenal tissue, prostate tissue, endometrial tissue,blood, skin, and breast tissue. Molecules of interest may be obtainedfrom a specific group of cells, such as tumor cells or fibroblast cells.Molecules of interest also may be obtained, and optionally partiallypurified or isolated, from the one or more biological samples usingstandard biochemical methods known in the art.

The frequency of biological sampling can vary depending on differentfactors. Such factors include, but are not limited to, the nature of themolecules of interest, ease and safety of sampling, synthesis andbreakdown/removal rates of the molecules of interest, and the half-lifeof an administered compound.

The molecules of interest may also be purified partially, or optionally,isolated, by any purification method known in the art including, but notlimited to, high pressure liquid chromatography (HPLC), fast performanceliquid chromatography (FPLC), chemical extraction, thin layerchromatography, gas chromatography, gel electrophoresis, and/or otherseparation methods known to those skilled in the art.

In another embodiment, the molecules of interest may be hydrolyzed orotherwise degraded to form smaller molecules. Hydrolysis methods includeany method known in the art, including, but not limited to, chemicalhydrolysis (such as acid hydrolysis) and biochemical hydrolysis (such aspeptidase degradation). Hydrolysis or degradation may be conductedeither before or after purification and/or isolation of the one or moremolecules of interest. The one or more molecules of interest also may bepartially purified, or optionally, isolated, by purification methodsincluding, but not limited to, high performance liquid chromatography(HPLC), fast performance liquid chromatography (FPLC), gaschromatography, gel electrophoresis, and/or any other methods ofseparating chemical and/or biochemical compounds known to those skilledin the art.

C. ANALYSIS

Conventional technologies (static methods) used to identify biologicalactions of compounds measure only composition, structure, orconcentrations of molecules in a cell and do so at one point in time. Incontrast, the methods of the present invention allow for the measurementof molecular flux rates of one or more molecules of interest in intactmetabolic pathways as described, infra.

1. Mass Spectrometry

Isotopic enrichment in one or more molecules of interest can bedetermined by various methods such as mass spectrometry, including butnot limited to gas chromatography-mass spectrometry (GC-MS),isotope-ratio mass spectrometry, GC-isotope ratio-combustion-MS,GC-isotope ratio-pyrrolysis-MS, liquid chromatography-MS, electrosprayionization-MS, matrix assisted laser desorption-time of flight-MS,Fourier-transform-ion-cyclotron-resonance-MS, and cycloidal-MS.

Mass spectrometers convert molecules into rapidly moving gaseous ionsand separate them on the basis of their mass-to-charge ratios. Thedistributions of isotopes or isotopologues of ions, or ion fragments,may thus be used to measure the isotopic enrichment in a plurality ofmolecules.

Generally, mass spectrometers include an ionization means and a massanalyzer. A number of different types of mass analyzers are known in theart. These include, but are not limited to, magnetic sector analyzers,electrospray ionization, quadrupoles, ion traps, time of flight massanalyzers, and Fourier transform analyzers.

Mass spectrometers may also include a number of different ionizationmethods. These include, but are not limited to, gas phase ionizationsources such as electron impact, chemical ionization, and fieldionization, as well as desorption sources, such as field desorption,fast atom bombardment, matrix assisted laser desorption/ionization, andsurface enhanced laser desorption/ionization.

In addition, two or more mass analyzers may be coupled (MS/MS) first toseparate precursor ions, then to separate and measure gas phase fragmentions. These instruments generate an initial series of ionic fragments ofa molecule, and then generate secondary fragments of the initial ions.

Different ionization methods are known in the art. One key advance hasbeen the development of techniques for ionization of large, non-volatilemacromolecules such as proteins and polynucleotides. Techniques of thistype have included electrospray ionization (ESI) and matrix assistedlaser desorption (MALDI). These have allowed MS to be applied incombination with powerful sample separation introduction techniques,such as liquid chromatography and capillary zone electrophoresis.

In addition, mass spectrometers may be coupled to separation means suchas gas chromatography (GC) and high performance liquid chromatography(HPLC). In gas-chromatography mass-spectrometry (GC/MS), capillarycolumns from a gas chromatograph are coupled directly to the massspectrometer, optionally using a jet separator. In such an application,the gas chromatography (GC) column separates sample components from thesample gas mixture and the separated components are ionized andchemically analyzed in the mass spectrometer.

When GC/MS (or other mass spectrometric modalities that analyze ions ofmolecules, rather than small inorganic gases) is used to measure massisotopomer abundances of molecules, hydrogen-labeled isotopeincorporation from isotope-labeled water is amplified 3 to 7-fold,depending on the number of hydrogen atoms incorporated into the moleculefrom isotope-labeled water in vivo.

In general, in order to determine a baseline mass isotopomer frequencydistribution for the molecule of interest, such a sample is taken beforeinfusion of an isotopically-labeled precursor. Such a measurement is onemeans of establishing in the cell, tissue or organism, the naturallyoccurring frequency of mass isotopomers of the molecule of interest.When a cell, tissue or organism is part of a population of subjectshaving similar environmental histories, a population isotopomerfrequency distribution may be used for such a background measurement.Additionally, such a baseline isotopomer frequency distribution may beestimated, using known average natural abundances of isotopes. Forexample, in nature, the natural abundance of ¹³C present in organiccarbon in 1.11%. Methods of determining such isotopomer frequencydistributions are discussed below. Typically, samples of the molecule ofinterest are taken prior to and following administration of anisotopically labeled precursor to the subject and analyzed forisotopomer frequency as described below.

a. Measuring Relative and Absolute Mass Isotopomer Abundances

Measured mass spectral peak heights, or alternatively, the areas underthe peaks, may be expressed as ratios toward the parent (zero massisotope) isotopomer. It is appreciated that any calculation means whichprovide relative and absolute values for the abundances of isotopomersin a sample may be used in describing such data, for the purposes of thepresent invention.

2. Calculating Labeled: Unlabeled Proportion of Molecules of Interest

The proportion of labeled and unlabeled molecules of interest is thencalculated. The practitioner first determines measured excess molarratios for isolated isotopomer species of a molecule. The practitionerthen compares measured internal pattern of excess ratios to thetheoretical patterns. Such theoretical patterns can be calculated usingthe binomial or multinomial distribution relationships as described inU.S. Pat. Nos. 5,338,686, 5,910,403, and 6,010,846, which are herebyincorporated by reference in their entirety. The calculations mayinclude Mass Isotopomer Distribution Analysis (MIDA). Variations of MassIsotopomer Distribution Analysis (MIDA) combinatorial algorithm arediscussed in a number of different sources known to one skilled in theart. The method is further discussed by Hellerstein and Neese (1999), aswell as Chinkes, et al. (1996), and Kelleher and Masterson (1992), andU.S. patent application Ser. No. 10/279,399, all of which are herebyincorporated by reference in their entirety.

In addition to the above-cited references, calculation softwareimplementing the method is publicly available from Professor MarcHellerstein, University of California, Berkeley.

The comparison of excess molar ratios to the theoretical patterns can becarried out using a table generated for a molecule of interest, orgraphically, using determined relationships. From these comparisons, avalue, such as the value p, is determined, which describes theprobability of mass isotopic enrichment of a subunit in a precursorsubunit pool. This enrichment is then used to determine a value, such asthe value A_(x)*, which describes the enrichment of newly synthesizedmolecules for each mass isotopomer, to reveal the isotopomer excessratio which would be expected to be present, if all isotopomers werenewly synthesized.

Fractional abundances are then calculated. Fractional abundances ofindividual isotopes (for elements) or mass isotopomers (for molecules)are the fraction of the total abundance represented by that particularisotope or mass isotopomer. This is distinguished from relativeabundance, wherein the most abundant species is given the value 100 andall other species are normalized relative to 100 and expressed aspercent relative abundance. For a mass isotopomer M_(X),

$\begin{matrix}{{{Fractional}\mspace{14mu} {abundance}\mspace{14mu} {of}\mspace{14mu} M_{X}} = A_{X}} \\{{= \frac{{Abundance}\mspace{14mu} M_{X}}{\sum\limits_{i = 0}^{n}{{Abundance}\mspace{14mu} M_{i}}}},}\end{matrix}$

where 0 to n is the range of nominal masses relative to the lowest mass(M₀) mass isotopomer in which abundances occur.

${{\Delta \mspace{14mu} {Fractional}\mspace{14mu} {abundance}\mspace{14mu} ( {{enrichment}\mspace{14mu} {or}\mspace{14mu} {depletion}} )} = {{( A_{x} )_{e} - ( A_{x} )_{b}} = {( \frac{{Abundance}\mspace{14mu} M_{X}}{\sum\limits_{i = 0}^{n}{{Abundance}\mspace{14mu} M_{i}}} )_{e} - ( \frac{{Abundance}\mspace{14mu} M_{X}}{\sum\limits_{i = 0}^{n}{{Abundance}\mspace{14mu} M_{i}}} )_{b}}}},$

where subscript a refers to enriched and b refers to baseline or naturalabundance.

In order to determine the fraction of polymers that were actually newlysynthesized during a period of precursor administration, the measuredexcess molar ratio (EM_(X)) is compared to the calculated enrichmentvalue, A_(X)*, which describes the enrichment of newly synthesizedbiopolymers for each mass isotopomer, to reveal the isotopomer excessratio which would be expected to be present, if all isotopomers werenewly synthesized.

3. Calculating Molecular Flux Rates

The method of determining rate of synthesis includes calculating theproportion of mass isotopically-labeled subunit present in the molecularprecursor pool, and using this proportion to calculate an expectedfrequency of a molecule of interest containing at least one massisotopically-labeled subunit. This expected frequency is then comparedto the actual, experimentally determined isotopomer frequency of themolecule of interest. From these values, the proportion of the moleculeof interest which is synthesized from added isotopically-labeledprecursors during a selected incorporation period can be determined.Thus, the rate of synthesis during such a time period is alsodetermined.

A precursor-product relationship then may be applied. For the continuouslabeling method, the isotopic enrichment is compared to asymptotic(i.e., maximal possible) enrichment and kinetic parameters (e.g.,synthesis rates) are calculated from precursor-product equations. Thefractional synthesis rate (k_(s)) may be determined by applying thecontinuous labeling, precursor-product formula:

k _(s)=[−ln(1−f)]/t,

where f=fractional synthesis=product enrichment/asymptoticprecursor/enrichment

and t=time of label administration of contacting in the living systemstudied.

For the discontinuous labeling method, the rate of decline in isotopeenrichment is calculated and the kinetic parameters of the one or moremolecules of interest are calculated from exponential decay equations.In practicing the method, biopolymers are enriched in mass isotopomers,preferably containing multiple mass isotopically-labeled precursors.These higher mass isotopomers of the molecules of interest, e.g.,molecules containing 3 or 4 mass isotopically labeled precursors, areformed in negligible amounts in the absence of exogenous precursor, dueto the relatively low abundance of natural mass isotopically-labeledprecursor, but are formed in significant amounts during the period ofmolecular precursor incorporation. The one or more molecules of interesttaken from the one or more cells, tissues, or organisms at thesequential time points are analyzed by mass spectrometry, to determinethe relative frequencies of a high mass isotopomer. Since the high massisotopomer is synthesized almost exclusively before the first timepoint, its decay between the two time points provides a direct measureof the rate of decay of the molecule of interest.

The first time point may be 2-3 hours after administration of precursorhas ceased, depending on mode of administration, to ensure that theproportion of mass isotopically-labeled subunit has decayedsubstantially from its highest level following precursor administration.In one embodiment, the following time points are typically 1-4 hoursafter the first time point, but this timing will depend upon thereplacement rate of the biopolymer pool.

The rate of decay of the molecule of interest is determined from thedecay curve for the three-isotope molecule of interest. In the presentcase, where the decay curve is defined by several time points, the decaykinetic can be determined by fitting the curve to an exponential decaycurve, and from this, determining a decay constant.

Breakdown rate constants (k_(d)) may be calculated based on anexponential or other kinetic decay curve:

k _(d)=[−ln f]/t.

As described, the method can be used to determine subunit poolcomposition and rates of synthesis and decay for substantially anybiopolymer which is formed from two or more identical subunits which canbe mass isotopically labeled. Other well-known calculation techniquesand experimental labeling or de-labeling approaches can be used (e.g.,see Wolfe, R. R. Radioactive and Stable Isotope Tracers in Biomedicine:Principles and Practice of Kinetic Analysis. John Wiley & Sons; (March1992)) for calculation flux rates of molecules and flux rates throughmetabolic pathways of interest.

4. Liquid Scintillation Counting

Radioactive isotopes may be observed using a liquid scintillationcounter. Radioactive isotopes such as ³H emit radiation that is detectedby a liquid scintillation detector. The detector converts the radiationinto an electrical signal, which is amplified. Accordingly, the numberof radioactive isotopes in a biological molecule may be measured.

In one embodiment, the radioisotope-enrichment value in a biologicalsample may be measured directly by liquid scintillation. In a furtherembodiment, the radio-isotope is ³H.

In another embodiment, the biological molecules or components thereofmay be partially purified, or optionally isolated, and subsequentlymeasured by liquid scintillation counting.

D. USES OF THE PRESENT INVENTION

The disclosed invention allows for systematic, high-throughput screeningof compounds or combinations of compounds or mixtures of compounds forunexpected, unintended therapeutic actions of drugs and unexpected drugtoxicities. FIG. 1 depicts the contemporary and traditional method ofdrug screening and drug development for intended actions in contrast tothe true importance of the full spectrum of unintended or unanticipatedevents in therapeutics and drug toxicity. FIG. 2 diagrams thehigh-throughput screening method for detecting new therapeuticindications elicited by known drug agents.

As these new indications have not been explicitly established previouslyand are not clearly predictable from pre-existing information, they areby definition unanticipated and unintended within context of the knowndrug's approved uses. The invention includes methods that use isotopekinetic techniques to measure the flux rates through metabolic pathwaysrelevant to disease. The discovery tools disclosed herein also allowvertical integration throughout the DDDA process—from preclinicaltesting through testing in human trials, which is distinct from currentmethods described in the art. The compound tested (or combinations ofcompounds or mixtures of compounds tested) can be already FDA-approveddrugs, thereby allowing discovery of new therapeutic indications andclaims for approved agents (see FIG. 2 and Table 4), or the compoundtested (or combinations of compounds or mixtures of compounds tested)can be any chemical entity or biological factor lacking FDA approval.

The current state of the art in pharmaceutical research and development,i.e., rational drug design coupled with target validation, allows for amore systematic approach to drug discovery than the earlier approach,used in the 1930's-1970's, of random screening of natural or syntheticchemical entities against crude physiologic models of disease. However,as can be seen in Table 1, rational drug design coupled with targetvalidation shares critical drawbacks with the earlier approach, notablya failure to detect unintended or unanticipated events such as secondarytherapeutic indications and/or toxic effects of compounds. This isbecause the contemporary DDDA approach is focused on narrowly targetedcriteria (such as HTS assays of enzyme targets or computer modeling ofenzyme active sites), and compounds such as already-approved drug agentsor new chemical entities or biological factors will unavoidably haveunintended or unpredictable actions on many other biologically importantprocesses outside the scope of the narrow target of interest. Theactivities of compounds, both therapeutic and toxic, are not restrictedto the intended targets that are screened and tested using contemporaryDDDA techniques simply because pharmacologists and physicians wished itwere so.

TABLE 1 Comparison of Old Drug Discovery (“Gifts from Nature”) to NewDrug Discovery (Rational) “Gifts from Rational Nature” Discovery Diseaseunderstood (pathogenesis)? No Yes Physiologic/Disease Model availableYes +/− for rapid screening? Chemical entities from Nature? Yes NoMolecular screening assay used No Yes Proven success? Yes +/− Chronicdiseases accessible to discovery? No Yes Unintended activities (toxic)identified? No No Unintended activities (secondary No No therapeutic)identified? Combination therapies (synergy, Yes No interactions)identified?

The methods of the present invention allow the researcher to rationallyscreen for unintended or unanticipated events elicited from compounds orcombinations of compounds or mixtures of compounds and to do so in ahigh-throughput fashion. For example, the methods disclosed herein allowthe study of relative molecular flux rates in hundreds or thousands ofanimals for periods of weeks or months. By using labeled water (e.g.,²H₂O) as the source of stable isotope, each animal (or human subject)drinks water containing labeled water, such as ²H₂O and this continuesfor as long as is required by the labeling protocol. If the molecularflux rates of two or more biological molecules are determined by massspectrometry, the method may be completely automated (autosampled,computerized data analysis and calculation of flux rates).

The methods disclosed herein enable the skilled artisan to measure themolecular flux rates of a variety of molecules of interest that comprisethe important metabolic pathways found in living systems. In manyinstances, altered flux rates in and through metabolic pathways can belinked to certain diseases, and measuring the flux rates of themolecules comprising these pathways will provide information useful indetecting secondary therapeutic indications and/or unintended toxiceffects of compounds or combinations of compounds or mixtures ofcompounds. Table 2 lists many of the molecules of interest comprisingmetabolic pathways in living systems:

TABLE 2 Some Biomolecules for Which Flux Rates (Synthesis and BreakdownRates; Input and Removal Rates), Can Be Measured for Screening ofChemical Entities for Actions in Biological Systems Class Examples I)Lipids and derivatives Acylglycerides Triglycerides PhospholipidsCardiolipin Fatty acids Palmitate Arachidonic acid Sterols CholesterolBile acids Estrogen, testosterone Glucocorticoids CeramidesSphingomyelin Galactocerebroside II) Carbohydrates and derivativesMonosaccharides Glucose Galactose Amino sugars N-Acetyl-GalactosaminePolysaccharides Glycogen Glycoproteins Sialic acid GlycolipidsGalactocerebrosides Glycosaminoglycans Hyaluronic acidChondroitin-sulfate Heparan-sulfate III) Proteins, peptides and aminoacids Structural proteins Collagen Myosin Secreted proteins AlbuminApolipoprotein B Insulin Immunoglobulins Prostate-specific antigenFibrinogen Interleukin-2 Secreted or excreted peptides N-terminalcollagen telopeptides Glutathione Pyridinolines Membrane proteinsPreadipocyte factor-1 Histocompatibility antigens T-cell receptorsModified amino acids Hydroxyproline 3-Methyl-histidine Intracellularproteins Creatine Enzymes Cytochrome C oxidase Transporters Glut-4Transcription factors PPAR-γ IV Nucleic acids DeoxyribonucleotidesGenomic DNA Mitochondrial DNA Viral or bacterial DNA RibonucleotidesMessenger RNA Ribosomal RNA Free nucleosides/nucleotides DeoxyadenosineDeoxythymidine Adenosine-triphosphate Purine and pyrimidine basesCytidine Adenine Metabolic products of bases Uric acid OligonucleotidesALU sequences 8-oxo-guanidine Methyl-deoxycytosine

The methods of the present invention allow for the high-throughputscreening of compounds or combinations of compounds or mixtures ofcompounds for actions in biological systems, particularly forunanticipated or unintended actions, by measuring the molecular fluxrates in metabolic pathways thought to be involved in diseases. In oneembodiment, the molecular flux rates in the one or more metabolicpathways of interest being measured may be relevant to an underlyingmolecular pathogenesis, or causation of, one or more diseases. Inanother embodiment, the molecular flux rates in one or more metabolicpathways of interest may contribute to the initiation, progression,severity, pathology, aggressiveness, grade, activity, disability,mortality, morbidity, disease sub-classification or other underlyingpathogenic or pathologic feature of the disease of interest.

In yet another embodiment, the molecular flux rates in one or moremetabolic pathways of interest may contribute to the prognosis,survival, morbidity, mortality, stage, therapeutic response,symptomology, disability or other clinical factor of the disease ofinterest. Two or more molecular flux rates in metabolic pathways may bemeasured independently or concurrently.

Such metabolic pathways may include, but are not limited to, hepatocyteproliferation and destruction, renal tubular cell turnover, lymphocyteturnover, spermatocyte turnover, protein synthesis and breakdown inmuscle and heart, liver collagen synthesis and breakdown, myelinsynthesis and breakdown in brain or peripheral nerves, breast epithelialcell proliferation, colon epithelial cell proliferation, prostateepithelial cell proliferation, ovarian epithelial cell proliferation,endometrial cell proliferation, bronchial epithelial cell proliferation,pancreatic epithelial cell proliferation, pancreatic beta cellregeneration, keratin synthesis in skin, keratinocyte proliferation,carbohydrate metabolism (including pathways affected by insulinresistance), cholesterol metabolism and clearance (including reversecholesterol transport), immunoglobulin synthesis, synthesis andbreakdown of mitochondrial DNA, synthesis and breakdown of mitochondrialphospholipids, synthesis and breakdown of mitochondrial proteins,synthesis and breakdown of adipose lipids, and synthesis and breakdownof adipose cells.

Known animal models of disease may be used as part of the presentinvention. Such animal models of disease may include, but are notlimited to, Alzheimer's disease, heart failure, renal disease, diabeticnephropathy, osteoporosis, hepatic fibrosis, cirrhosis, hepatocellularnecrosis, pulmonary fibrosis, scleroderma, renal fibrosis, multiplesclerosis, arteriosclerosis, osteoarthritis, rheumatoid arthritis,psoriasis, skin photoaging, skin rashes, breast cancer, prostate cancer,colon cancer, pancreatic cancer, lung cancer, acquired immunodeficiencysyndrome, immune defects, multiple myeloma, chronic lymphocyticleukemia, chronic myelocytic leukemia, diabetes, diabetic complications,insulin resistance, obesity, lipodystrophy, metabolic syndrome, musclewasting, frailty, deconditioning, angiogenesis, hyperlipidemia,infertility, viral or bacterial infections, auto-immune disorders, andimmune flares

Table 3 contains exemplary examples of metabolic pathways and associateddisease states. Although extensive, the list contained in Table 3 is notintended to be limiting, as one of skill would understand that themethods of the present invention are useful for any disease whoseetiology is at least in part defined by one or more altered flux ratesin one or more metabolic pathways.

TABLE 3 Examples of Metabolic Pathways that Can Be Measured (andRelevant Diseases) to Screen for Unanticipated or Unintended Actions inBiological Systems Metabolic Pathway Disease I) DNA replication (celldivision) Hepatocytes Hepatitis; hepatic necrosis Lymphocytes (includingantigen- AIDS; vaccination specific T-cells) Spermatocytes Maleinfertility Colonocytes Colon cancer and colitis Mammary epithelialcells Breast cancer Renal tubular cells Nephrotoxins Prostate epithelialcells Prostate cancer; BPH Tumor cells Cancer, leukemia Vascular smoothmuscle cells Atherosclerosis Mitochondria Metabolic fitness;mitochondrial diseases Pancreatic β-cells Type 1 diabetes Bone marrowprogenitor cells Bone marrow failure Keratinocytes Psoriasis Endometrialcells Endometrial cancer Endothelial cells Angiogenesis II) Fibrogenesisand bone deposition Liver collagen synthesis Liver fibrosis; cirrhosisLung collagen synthesis Pulmonary fibrosis Cardiac collagen synthesisHeart failure Renal collagen synthesis Renal fibrosis Dermal collagensynthesis Scleroderma Bone collagen synthesis Osteoporosis; Paget'sDisease Cartilage collagen synthesis Osteoarthritis III) Lipid synthesisand breakdown Adipose tissue triglycerides Obesity; Lipodystrophy Serumcholesterol Hyperlipidemia Brain myelination and demyelination MultipleSclerosis Mitochondrial phospholipids Metabolic fitness Sterols Gallbladder disease; dyslipidemia; hormonal disorders IV) Tissueglycosaminoglycans Synovial fluid hyaluronic acid Osteoarthritis;rheumatoid arthritis Synovial fluid chondroitin-sulfate Osteoarthritis;rheumatoid arthritis Cartilage hyaluronic acid and Osteoarthritis;rheumatoid arthritis chondroitin-sulfate Tumor hyaluronic acidMetastatic potential V) Protein synthesis (general) ImmunoglobulinsMultiple myeloma; vaccination Albumin Malnutrition Apolipoprotein B or EHyperlipidemia Muscle myosin Frailty Skin keratin Psoriasis Amyloid-βAlzheimer's disease Viral or bacterial proteins Infectious diseasesInsulin Diabetes mellitus Interleukins and cytokines Inflammation Hairproteins Hirsutism; baldness Histocompatability proteins TransplantationHemoglobin Anemias Histones Gene regulation Fibrinogen Clottingdisorders VI) Carbohydrate synthesis Blood glucose Diabetes mellitusGalacto-cerebrosides Multiple Sclerosis Advanced glycosylation productsDiabetic complications Tissue glycogen Insulin resistance

The methods of the present invention allow for the identification ofsecondary indications for FDA (or other corresponding agencies outsideof the U.S.)-approved drugs. With regard to secondary (unintended)therapeutic actions of drugs, it should be noted that, once a drug isapproved by the FDA, (or other corresponding agencies outside the U.S.)it may be prescribed by a physician for purposes other than theFDA-approved (i.e., intended and rigorously tested) indication orindications. Indeed, “off-label” use accounts for the majority (>60%) ofprescriptions written in the U.S. Such off-label use often accounts forthe greatest commercial and medical impact of approved drugs (Table 2,supra). Failure to recognize or be aware of such secondary (i.e.,unintended or unanticipated) therapeutic actions has often preventedpharmaceutical companies from obtaining the full commercial advantage oftheir proprietary agents before expiration of patent protection.Therefore, new uses for approved agents represents one of the mostattractive and commercially profitable approaches for developing newtherapeutic agents in the United States, because approved agents havethe largest human experience relative to potential toxicities, havealready undergone full safety and dosing testing, and are familiar tomedical providers.

Known or approved drug agents may be screened for actions on single ormultiple metabolic pathways independently or concurrently. The drugs maybe selected randomly or the drugs may be selected on the basis of aspecific biochemical rationale or hypothesis concerning a hypothesizedrole in the molecular pathogenesis of one or more diseases. Non-approveddrugs, such as new chemical entities, non-approved biological factors,drug leads (including biological leads), and the like also may beselected randomly or may be selected on the basis of a specificbiochemical rationale or hypothesis concerning a hypothesized role inthe molecular pathogenesis of one or more diseases.

Such known or approved drugs may include, but are not limited to,statins, glitazones, COX-2 inhibitors, NSAIDS, Ji-blockers, calciumchannel blockers, ACE inhibitors, antibiotics, antiviral agents,hypolipidemic agents, antihypertensives, anti-inflammatory agents,antidepressants, anxiolytics, anti-psychotics, sedatives, analgesics,antihistamines, oral hypoglycemic agents, antispasmodics,antineoplastics, cancer chemotherapeutic agents, sex steroids, pituitaryhormones, cytokines, chemokines, appetite suppressant agents,thyromimetics, anti-seizure agents, sympathomimetics, sulfa drugs,biguanides, and other classes of agents. Indeed, there are numerousexamples of secondary claims or off-label uses of approved drugs in thecurrent therapeutic armamentarium (Table 4).

TABLE 4 Examples of Secondary Indications or Off-Label Uses of ApprovedDrugs Minoxidil (hair loss) Metformin (PCO-NASH) Quinidine (malariapatients w/A-Fib improved) Chloroquine (malaria −> RA + SLE) Carbonicanhydride inhibitors (sulfanilamide derivative) Sulfonylureas (sulfa's−> hypoglycemia) Prazosin (BPH) Bisphosphonates Megace (weight gain incachexia) β-agonists (muscle gain) Retinoids (wrinkles) Tegretol +Elavil (DM neuropathy) MAO's (INH antidepressant activities) ACEinhibitors (DM nephropathy; CHF fibrosis) COX-2 (CLL; colonchemoprevention) Sulfasaline (Crohn's disease) Thalidomide (weight gain;apthous ulcers) Statins (osteoporosis/Alzheimer's disease; multiplesclerosis) Amiodarone (angina −> arrhythmias) Chlorpropamide (DI)Amantadine (Parkinson's disease) Niacin (HLP) Methotrexate (RA) PTU(cirrhosis) β-blockers/Ca⁺⁺ blockers (migraine) Tegretol(manic-depression) Dilantin (trigeminal neuralgia) Diazoxide(insulinoma)

Combination therapies represent another area that the contemporary DDDAsystem misses entirely. Drugs may act synergistically on a diseaseprocess by acting on different steps in the disease pathway (FIG. 2).These interactions will not be observable through screening proceduresthat measure one targeted enzyme or gene at a time. Because drugcombinations are themselves patentable and can provide an additionalperiod of exclusivity to a pharmaceutical company, the failure toidentify effective, unanticipated drug combinations represents a hugecommercial loss to the industry. The methods of this invention allowcombination therapies to be systematically identified by ahigh-throughput screening approach (Table 5).

TABLE 5 Examples of Combination Therapies Cancer chemotherapies Leukemiachemotherapies HIV/AIDS (e.g., proteaseinhibitor/nucleoside/non-nucleoside reverse transcriptase inhibitorcocktails) Bacterial infection (e.g., sulfonamide/trimethoprin) Diabetes(e.g., bed-time insulin/day-time sulfonyl urea) Anti-hypertensives(e.g., calcium-channel blockers/thiazole diuretics) Heart disease (e.g.,beta-blockers/nitrates for angina; digoxin/beta- blockers for congestiveheart failure)Agents with different sites of action in the same pathway may havecomplementary, synergistic, or antagonistic effects on flux ratesthrough the pathway of interest in vivo (e.g., flux rates in de novo DNAsynthesis: see FIG. 3).

These interactions between agents cannot be detected or quantified byuse of contemporary or traditional assays that investigate one moleculartarget and step at a time in a disease-related pathway. Many examples ofextremely useful combination drug therapies have emerged in medicaltherapeutics (see Table 5), generally by serendipitous discovery,trial-and-error in humans with a disease, or based on theoreticalbiochemical interactions. A method for systematically screening andevaluating combinations of agents for effects on fluxes throughpathways, particularly for screening and evaluating known, approveddrugs, had not previously been available. The invention disclosed hereinwould facilitate the process of identifying, developing and approvingeffective therapeutic combinations.

In another embodiment, the methods of the invention are useful indetecting toxic effects of compounds. Unanticipated toxicities ofcompounds often emerge in phase II-III FDA clinical trials or, evenworse, during post-approval clinical use of a drug. Such toxicities arecommon and represent an enormous source of financial losses to thepharmaceutical industry. The failure of the DDDA system to identify mosttoxicities early in the DDDA process or to monitor for their occurrenceprior to clinical signs and symptoms represents a basic failure of thecurrent DDDA process that has an adverse impact on public health as wellas pharmaceutical company commercial profitability. Such toxic effects(unanticipated toxicities) may include end-organ toxicity. End-organtoxicity may include, but is not limited by, hepatocyte proliferationand destruction, renal tubular cell turnover, lymphocyte turnover,spermatocyte turnover, protein synthesis and breakdown in muscle andheart, liver collagen synthesis and breakdown, myelin synthesis andbreakdown in brain or peripheral nerves, breast epithelial cellproliferation, colon epithelial cell proliferation, prostate epithelialcell proliferation, ovarian epithelial cell proliferation, endometrialcell proliferation, bronchial epithelial cell proliferation, pancreaticepithelial cell proliferation, keratin synthesis in skin, keratinocyteproliferation, immunoglobulin synthesis, synthesis and breakdown ofmitochondrial DNA, synthesis and breakdown of mitochondrialphospholipids, synthesis and breakdown of mitochondrial proteins,synthesis and breakdown of adipose lipids, and synthesis and breakdownof adipose cells.

E. ISOTOPICALLY-PERTURBED MOLECULES

In another variation, the methods provide for the production of isolatedisotopically-perturbed molecules (e.g., labeled fatty acids, lipids,carbohydrates, proteins, nucleic acids and the like). These isolatedisotopically-perturbed molecules comprise information useful indetermining the flux of molecules within the metabolic pathways ofinterest. Once isolated from a cell and/or a tissue of an organism, oneor more isolated isotopically-perturbed molecules are analyzed toextract information as described, supra.

F. KITS

The invention also provides kits for measuring and comparing molecularflux rates in vivo. The kits may include isotope-labeled precursormolecules, and may additionally include chemical compounds known in theart for separating, purifying, or isolating proteins, and/or chemicalsnecessary to obtain a tissue sample, automated calculation software forcombinatorial analysis, and instructions for use of the kit.

Other kit components, such as tools for administration of water (e.g.,measuring cup, needles, syringes, pipettes, IV tubing), may optionallybe provided in the kit. Similarly, instruments for obtaining samplesfrom the cell, tissue, or organism (e.g., specimen cups, needles,syringes, and tissue sampling devices) also may be optionally provided.

G. INFORMATION STORAGE DEVICES

The invention also provides for information storage devices such aspaper reports or data storage devices comprising data collected from themethods of the present invention. An information storage deviceincludes, but is not limited to, written reports on paper or similartangible medium, written reports on plastic transparency sheets ormicrofiche, and data stored on optical or magnetic media (e.g., compactdiscs, digital video discs, magnetic discs, and the like), or computersstoring the information whether temporarily or permanently. The data maybe at least partially contained within a computer and may be in the formof an electronic mail message or attached to an electronic mail messageas a separate electronic file. The data within the information storagedevices may be “raw” (i.e., collected but unanalyzed), partiallyanalyzed, or completely analyzed. Data analysis may be by way ofcomputer or some other automated device or may be done manually. Theinformation storage device may be used to download the data onto aseparate data storage system (e.g., computer, hand-held computer, andthe like) for further analysis or for display or both. Alternatively,the data within the information storage device may be printed ontopaper, plastic transparency sheets, or other similar tangible medium forfurther analysis or for display or both.

H. EXAMPLES

The following non-limiting examples further illustrate the inventiondisclosed herein and one of skill in the art will appreciate thatvariations in the procedures described in the Examples, supra, and readin light of what is disclosed in the specification, are fullyencompassed by the invention as claimed:

Example 1 Measurement of Triglyceride (TG) Synthesis (Lipogenesis) andBreakdown (Lipolysis) in Rats after Exposure to Compounds

To assess whether a compound inhibits lipogenesis (and therefore, acandidate drug for treating obesity or other metabolic disorders)Sprague-Dawley rats (200-300 g Simonsen Labs, Gilroy, Calif.) are eitherexposed to a compound or left unexposed (i.e., controls). Rats areadministered compound or vehicle via gavage. One or several compoundsmay be administered. For example, thousands of compounds may beinitially screened, pooled, rescreened, subpooled, etc., to screen forone or more active compounds. An initial priming dose of 99.8% ²H₂O isgiven via intraperitoneal injection to achieve ca. 2.5% body waterenrichment (assuming 60% body weight as water) followed byadministration of 4% ²H₂O in drinking water for up to 12 weeks.

Adipose tissue samples are placed in dual glass tissue grinders (e.g.,Kontes tissue grinders, Kimble Kontes, Vineland, N.J.) with 1 mlmethanol:chloroform (2:1), ground until homogenous then centrifuged toremove protein. The solution is extracted with 2 ml each chloroform andwater. The aqueous phase is discarded and the lipid fraction istransesterified by incubation with 3N methanolic HCL (Sigma-Aldrich) at55° C. for 60 min. Fatty acid methyl esters are separated from glycerolby the Folch technique, with the modification that pure water ratherthan 5% NaCl is used for the aqueous phase. The aqueous phase containingglycerol is then lyophilized and glycerol is converted to glyceroltri-acetate by incubation with acetic anhydride:pyridine, 2:1 asdescribed elsewhere (Hellerstein, M. K., R. A. Neese, and J. M. Schwarz.Am J Physiol 265:E814-20, 1993, herein incorporated by reference). Somesamples are extracted and then TG separated from other acylglycerides bythin layer chromatography (TLC) as described elsewhere (Jung, H. R., S.M. Turner, R. A. Neese, S. G. Young, and M. K. Hellerstein. Biochem 3343Pt 2:473-8, 1999, herein incorporated by reference), then analyzed asdescribed, supra.

Glycerol-triacetate is analyzed for isotope enrichment by GC/MS, asdescribed, supra.

The fraction of TG that is newly synthesized, (f) is calculated asdescribed, supra.

The theoretical plateau or asymptotic value (A₁ ^(∞)) in TG-glycerolduring ²H₂O labeling is determined in two ways: by mass isotopomerdistribution analysis (MIDA) of the combinatorial labeling pattern inglycerol (A₁ ^(∞) _(mida)) and by measurement of plateau enrichmentsreached in “fully replaced” TG depots (A₁ ^(∞) plateau) (see below). Thestandard precursor-product equation is then applied:

f=1−e−ks*t

ks=−ln(1−f)/t

Where ks represents the fractional replacement or synthesis rateconstant and t is time of labeling.

The absolute synthesis rate of adipose TG is calculated by multiplyingthe measured fractional synthesis (ks) over the period of labeling timesthe pool size of TG. For the purpose of this calculation, TG content isassumed to be 10% of body weight in non-obese young rodents. Theabsolute synthesis rate of adipose tissue TG can be calculated asfollows,

Absolute synthesis(mg/d)=ks(d−1)×TG content(mg)

For statistical analysis, ANOVA is used to compare groups with p<0.05 asthe criteria for significance. Curve fitting of label incorporation datais performed using Delta Graph (Delta Point, Inc.).

TG synthesis rates are then compared between exposed animals andunexposed animals to determine whether a compound (or combination ofcompounds or mixture of compounds) inhibits lipogenesis

One can also assess whether a compound (or combination of compounds ormixture of compounds) stimulates lipolysis using the same protocols asdescribed above. The net lipolytic (TG breakdown) rate in individual fatdepots is calculated from the difference between the absolute rate of TGsynthesis and the net rate of TG accumulation, where the latter isdetermined from the change in weight over time in a fat pad or in thewhole body:

Net lipolysis(mg/d)=Absolute TG synthesis−net TGaccumulation=([ks(d−1)×TG content(mg)]−[(change in TG content)/time(d)]

Compound-exposed animals are then compared to unexposed animals todetermine if the compound (or combination of compounds or mixture ofcompounds) has lipolytic activity.

Example 2 Measurement of DNA Synthesis and Breakdown in Rats afterExposure to Compounds

DNA synthesis is a biomarker for cell proliferation. In some settings itmay be desirable to stimulate cell proliferation (e.g., to stimulatewound healing) while in other settings it may be desirable to inhibitcell proliferation (e.g., cancer).

Rats are administered ²H₂O as discussed in Example 1, supra.

Rats are either administered compounds (or combinations of compounds ormixtures of compounds) or vehicle (controls) as discussed in Example 1,supra.

DNA is then isolated from the tissue or cell of interest using a Qiagenkit (Qiagen, Valencia, Calif.), following the manufacturer's protocol.

Isotope enrichment is then analyzed and flux rates calculated asdescribed, supra. DNA synthesis is then determined as described, supra,(and in U.S. Pat. No. 5,910,403, incorporated by reference).Compound-exposed animals are then compared to unexposed animals todetermine if the compound (or combination of compounds or mixture ofcompounds) has an effect on DNA synthesis.

Example 3 Measurement of Neurogenesis in Rat Hippocampal NeuroprogenitorCells after Exposure to Compounds

Compounds (or combinations of compounds or mixtures of compounds) aretested on rats to determine whether one or more may have effects onneurogenesis. Compounds with neurogenic potential may find use intreating spinal cord injury, Parkinson's disease, Huntington's diseaseand other neurodegenerative disorders. Rats are divided into exposed andcontrol groups and administered labeled water as in Example 1, supra.After exposure to compound or combinations of compounds or mixtures ofcompounds (or vehicle if control rat), by gavage, intrathecal, orintracranial administration (route of administration is dependent on thechemistry of the compound or combination of compounds or mixture ofcompounds, as is well known in the art) rats are deeply anesthetizedwith a mixture of ketamine, xylazine, and acepromazine. Rats are thendecapitated and whole brains are removed.

For isolating tissue for neurogenesis analysis, the brain is bisectedlongitudinally and each hippocampal lobe is separated from theoverlaying cortical white matter using the natural separation line alongthe alveus hippocampus. The white matter of the Fimbria and subiculumisremoved.

Tissues are finely minced and digested for 45 min in a solution ofpapain (2.5 U/ml; Worthington, Freehold, N.J.), DNase (250 U/ml,Worthington), and neutral protease (1 U/ml Dispase; Boehringer Mannheim,Indianapolis, Ind.) dissolved in HBSS. (Alternatively, tissue can alsobe digested for 45 min in DMEM containing a mixture of 0.1% papain and0.01% DNase).

Cells and tissue fragments are washed three times with DMEM containing10% FBS (Hyclone, Logan, Utah).

Whole digested tissue is then suspended in DMEM-10% FBS, filteredthrough a sterile 107 μM nylon mesh and thoroughly mixed with an equalvolume of Percoll solution. The Percoll solution is made by mixing nineparts of Percoll (Amersham Pharmacia Biotech, Uppsala, Sweden) with onepart 10×PBS (Irvine Scientific, Santa Ana, Calif.).

The cell suspension is then fractionated by centrifugation for 30 min,18° C., at 20,000×g. Cell fractions are harvested and washed free ofPercoll by three or more rinses in DMEM-10% FBS.

DNA synthesis is measured as in Example 2, supra.

Compound-exposed animals are then compared to unexposed animals todetermine if the compound (or combination of compounds or mixture ofcompounds) has an effect on DNA synthesis in hippocampal neuroprogenitorcells.

Example 4 Measurement of Flux Rates of Alzheimer Disease-RelatedProteins From Mice after Exposure to Compounds

Mice are labeled with ²H₂O using the procedures described in Example 1,supra, for rats. Mice are given compound via gavage, intrathecal, orintracranial administration. Urine is collected to isolate amyloid beta(Aβ) protein. Total urinary protein is concentrated and exchanged in asuitable buffer for immunoaffinity purification. After immunoaffinitypurification, Aβ can be further purified using size exclusion and/orreversed phase chromatography. The identity of purified peptides isconfirmed by ELISA, western blot, and LC-MS (ESI).

Mice are sacrificed and brain tissue is extracted and amyloid precursorprotein (APP) and C-terminal fragment of APP(CTF) are obtained. Brainproteins are extracted in neutral buffer, insoluble material is removed,and proteins precipitated. Resulting material is exchanged into an ionexchange buffer, and purified by ion exchange chromatography and thensize exclusion and/or reversed phase chromatography. The identity ofpurified protein is confirmed by ELISA and western blot.

Enrichments for Aβ, APP, and CTF are performed as described, supra.Molecular flux rates for Aβ, APP, and CTF are calculated as described,supra. Compound-exposed animals are then compared to unexposed animalsto determine if the compound (or combination of compounds or mixture ofcompounds) has an effect on AD, APP, and CIF synthesis or degradation.

Example 5 Glycolytic Disposal of Glucose in Normal Rats after Exposureto Compounds

Glycolytic disposal is a biomarker for insulin resistance and type IIdiabetes (see Reaven G M. Banting Lecture 1988. Role of insulinresistance in human disease. Diabetes 37(12):1595-607, 1988). Rats, asin Example 1, supra, are used to measure glycolytic disposal in vivo inresponse to compounds (or combinations of compounds or mixtures ofcompounds).

The ²H-glucose labeling protocol consists of an initial intraperitoneal(ip) injection of 99.9% [6,6-²H₂] glucose. For labeling rats, 2 mglabeled glucose per gram body weight is introduced. Body water iscollected as serum at various timepoints. Compounds (or combinations ofcompounds or mixtures of compounds) are administered by gavage.

Glycolysis is measured by measuring deuterium in body water as a percentof administered [6,6-²H₂] glucose normalized to account for differentmolar quantities of deuterium in molecular glucose and molecular water.Deuterated water is measured as described, supra. Glycolysis fromdrug-exposed rats is compared with glycolysis from unexposed rats todetermine if any compound (or combination of compounds or mixture ofcompounds) had an effect on glycolysis.

Example 6 Brain Galactocerebroside Turnover as a Measurement ofRemyelination after Exposure of Compounds

Rats are given deuterated water as in Example 1, supra. Rats areadministered compounds (i.e., compounds not approved for inhibition ofdemyelination and/or stimulation of remyelination) via gavage,intrathecal, or intracranial administration. Demyelination is animportant biomarker for multiple sclerosis (MS). Remyelination mayindicate a potential drug candidate for treating MS.

Weigh a set of 2-mL microcentrifuge tubes. Brains are collected from ratcarcasses and put it into the pre-weighed microcentrifuge tubes. Themicrocentrifuge tubes are weighed again. The net weight is the brainweight. The brain is put onto an ice-cooled glass plate, and 10 crystalsof BHT are added. A razor blade is used to mince the brain for 1 minute.A spatula is used to put the minced brain back into the microcentrifugetubes. The brain is minced well with a spatula. 80-120 mg of mincedbrain is put into 13×100 mm glass tubes with PTFE screw caps ensuringthe tissue is at the bottom of the tube. The rest of the brain is storedin the microcentrifuge tubes at −20° C. 2 mL of chloroform-methanol 2:1(v/v) with BHT is added into the glass tubes and the tubes are vortexedensuring that all of the tissues are soaked in the solvent. Stand 3 h atroom temperature in a dark area. The caps are taken off the glass tubes.The tubes are centrifuged at 2000 RCF for 10 minutes at roomtemperature. The supernatant (lipid extracts) is poured into 2-mL screwcapped vials and the solid residue is discarded.

100 mL of developing solvent (chloroform-methanol-water:69.15%:26.60%:4.26%) is added into the TLC separation tanks 1 h beforeadding the TLC plates. A20 mL pipette is used to spot 20 mL of totalcerebroside standard on lanes 1, 10, 19 of Whatman LK6DF silica gel 60TLC plates. For each sample, a 20 mL pipette is used to spot 100 μL oflipid extracts on two neighboring lanes (50 mL/lane). Wait until TLCplates look visually dry. The TLC plates are developed in the developingtanks. Each tank holds two plates, facing each other. Normally it takes40-45 minutes for the plates to be fully developed. After TLC platesdevelop, wait 15 minutes for the plates to dry. 20 iodine crystals areput into a tank specially used for iodine vapor. The tank is put on aheatblock set at 80° C. The dried TLC plates are put in the iodine tankto visualize the spots of lipids containing double bonds. The spots oftotal cerebroside standard are matched with those of samples. The TLCplate images are scanned by a computer. The silica gel is collected ontoa weighing box and transfered to a 12×75 mm disposable glass tube. 1 mLof chloroform-methanol 2:1 is added with BHT and vortexed. Let standuntil silica settles. The solvent is poured into a 13×100 mm screw captube. The solid residue is discarded. 1 mL of 3 N methanolic HCl isadded into the tube and the tube is capped tightly. The tubes are put ona heatblock at 80° C. for 2 h. The tubes are then removed from theheating block and allowed to cool to room temperature. 1.5 mL H2O and 3mL hexane are added into the tubes and the tubes are vortexed. 1.8-2 mLof the bottom layer (methyl glucose and methyl galactose) aretransferred to GC vials. The GC vials are put into a fitted rotor of theJouan 10.10 speedvac and the rotor is balanced and set at 60° C. Vacuumuntil the tubes are dry. 100 μL of acetic anhydride-pyridine 2:1 (v/v)is added to the GC vials and the vials are covered and allowed to standfor 1 h at room temperature. The vials are then blown down under N2until dry. 100 μl ethyl acetate is added and the vials are vortexed. Themixture is transferred to GC inserts and the vials are capped with acram per. The samples are run on the GC/MS and galactocerebrosideenrichments are determined. The molecular flux rates ofgalactocerebroside is determined as described supra, from rats exposedto compounds (or combinations of compounds or mixtures of compounds) andunexposed (vehicle control) rats. Enrichments of galactocerebrosidegreater than galactocerebroside enrichments in control rats indicatesincreases synthesis of galactocerebroside and possible remyelination.Enrichments that are less than controls indicates demyelination.

Example 7 Unanticioated Neurogenesis after Exposure toLipopolysaccharide (LPS)

Groups of mice (n=5/group) were given a bolus of ²H₂O (Spectra Isotopes,Columbia, Md.) to 5% ²H₂O in body water and were given 8% ²H₂O asdrinking water. Mice were maintained on ²H₂O for 45 days, during whichperiod they also received injections every other day of eitherlipopolysaccharide (LPS) (Sigma, St. Louis, Mo.) in saline at a dose of1 or 4 mg/kg body weight, or no injection.

Mice were euthanized with carbon dioxide, and their brains were removed.Hippocampi were dissected out, trypsinized, and stained with tetanustoxoid (Ttx), a specific marker for neurons, and then stained with aFITC labeled anti-Ttx antibody (Roche, Basel Switzerland). Ttx and PI(DNA) positive cells were isolated on a cell sorter (Coulter EPICSElite), and isolated cells were run through a commercially available kit(DNeasy tissue kit, Qiagen, Valencia, Calif.) to isolate their DNA. DNAwas subsequently hydrolyzed, derivatized, and analyzed as described,supra.

Neurons were seen to turnover more rapidly in response to LPS in a dosedependent manner (see FIG. 4). LPS given as described is known to induceneuroinflammation in rodents. Previous work with neuroinflammatorystimuli using different, less sensitive techniques has shown a potentiallink between neuroinflammation and the suppression of neurogenesis. Withthis in mind, the Applicant had expected to observe the neurons to turnover more slowly since chronic exposure of neuroprogenitor cells to LPSwould be expected to adversely effect the viability of neuroprogenitorcells and thereby decrease neurogenesis, as is well known in the art(see, e.g., Ekdahl C. T. et al. Proc. Natl. Acad. Sci. 2003100(23):13632-13637, wherein neurogenesis was observed to decrease inthe presence of chronic exposure to LPS). As can be seen in FIG. 5, theexact opposite was observed. In particular, the calculated fractionalsynthesis rate (f) of new neurons nearly doubled (from approximately 7%to nearly 14° k) after exposure to LPS (see FIG. 4), when a reduction inf was the expected result. Since increases in neurogenesis arecorrelated with anti-depressant agents (e.g., fluoxetine), these resultssuggest that LPS may alleviate depression, a previously unknownindication of LPS.

Example 8 Unanticipated Decrease in Adipocyte Proliferation in ob/obMice after Exposure to Leptin

Four week old, female, C57/b16j mice (Jackson Labs Bar Harbor, Mich.)were studied: C57/bl6j^(+/?) controls (eon), ad libitum fedC57/bl6j^(lep−/lep−) (ob/ob), leptin treated ob/ob (ob-lep) and foodrestricted ob/ob (ob-r). Mice were housed individually in hanging wirecages and fed AIN 93 purified diets (Bio Serv, Frenchtown, N.J.). Micewere given 3 days to acclimate to the environment, after which mice grewnormally. All treatments and interventions began 5 days prior to thestart of labeling with ²H₂O.

The food intake of the ob-r group was restricted and administered in acontinuous manner with automatic pellet dispensers (CoulbournInstruments, Allentown Pa.).

Ob-lep mice received murine leptin subcutaneously at a dose of 2 μg/day(Amgen, Thousand Oaks Calif.) via a 28 day Alzet mini osmotic pump (AlzaCorp. Palo Alto Calif.).

Mice were injected with ²H₂O (deuterated water) 0.012 ml/gm. The normaldrinking water was then replaced with water enriched to 4% ²H₂O. ²H₂Otreatment had no impact on food intake or body weight. Twenty-one daysfollowing the start of ²H₂O administration, mice were fasted for fourhours, anesthetized with isoflurane and exsanguinated via heartpuncture.

Adipose Tissue Preparation and Isolation of Adipocytes

Fat pads were isolated and dissected inguinal and retroperitoneal pads,the left and right sides were pooled for analysis.

Immediately following dissection, the fat pads were placed in HBSS withcalcium in pre weighed tubes for isolation of mature adipocytesaccording to the method of Rodbell (Rodbell, M. Metabolism of isolatedfat cells. Effects of hormones on glucose metabolism and lipolysis. J.Biol. Chem. 239, 375-380 (1964)). Minced tissue was placed in HBSS with0.1% Type II collagenase (Worthington). Tissue was incubated at 37° forup to 90 minutes. Samples were spun at 800 rpm for 10 minutes. Theadipose cell enriched fraction was carefully removed from the middlefraction and frozen.

DNA Isolation and Derivatization from Adipose Cells:

The frozen slurry of adipocytes was lyophilized, the dry weight of thesample was determined and then the samples were digested and DNAisolated as described elsewhere (Neese, R. A. et al. Advances in theStable Isotope-Mass Spectrometric Measurement of DNA Synthesis and CellProliferation. Anal Biochem 298, 189-195. (2001)) using Quiagen DNeasytissue kits. The yield of DNA from each sample was determined with aPharmacia Biotec Genequant II spectrophotometer.

Ten to 25 μg of DNA was hydrolyzed to individual ribonucleic acids asdescribed in detail elsewhere (Neese, R. A. et al. Advances in theStable Isotope-Mass Spectrometric Measurement of DNA Synthesis and CellProliferation. Anal Biochem 298, 189-195. (2001)). Isolateddeoxyadenosine reduced and acetylated as described previously (Neese, R.A. et al. Advances in the Stable Isotope-Mass Spectrometric Measurementof DNA Synthesis and Cell Proliferation. Anal Biochem 298, 189-195.(2001)). The resulting pentose-tetraacetate (PTA) derivative in ethylacetate was injected into the GC/MS for measurement of isotopeenrichments of the deoxyribose moiety of DNA.

Derivatization and Analysis of H₂O:

²H₂O enrichments in body water were measured in tetrabromoethylenederivatized from plasma samples as described in detail elsewhere (Neese,R. A. et al. Advances in the Stable Isotope-Mass SpectrometricMeasurement of DNA Synthesis and Cell Proliferation. Anal Biochem 298,189-195. (2001).

GC/MS Analyses:

Model 5970 and 5971 GC/MS or 5973 instruments (Agilent, Palo Alto,Calif.) were used for measuring isotopic enrichments ofglycerol-triacetate fatty acid-methyl esters and tetrabromoethylene

Tetrabromoacetylene was analyzed using a DB-225 fused silica column,monitoring m/z 265 and 266 (parent M0 and M1 masses). Standard curves ofknown ²H₂O enrichment were run before and after each group of samples tocalculate isotope enrichment.

PTA samples were analyzed for incorporation of deuterium on a HP model5973 MS with a 6890 GC and auto-sampler (Agilent, Palo Alto, Calif.).Methane CI was used with a 30 m DB-225 column under selected ionmonitoring of m/z 245-246 (representing the M0 and M1 masses). Naturalabundance, (unenriched) dA samples were measured concurrently and theexcess M1 (EM1) abundance in the adipose PTA samples were calculated bydifference (subtraction of the M1 abundance measured in the unenrichedstandard from the M1 abundance in the sample). Bone marrow DNA sampleswere run simultaneously and used to represent a completely ornear-completely turned over tissue for calculating fractional adiposecell replacement, as described previously (Neese, R. A. et al. Advancesin the Stable Isotope-Mass Spectrometric Measurement of DNA Synthesisand Cell Proliferation. Anal Biochem 298, 189-195. (2001)).

Calculations

(see FIG. 5).

Example 9 Unanticipated Increase in Liver Cell Proliferation in Miceafter Exposure to Low Dose of Griseofulvin

Mice were given ²H₂O (as described in Example 7, supra, except that micewere maintained on deuterated water for 5 days, the duration of theexperiment) and divided into a control group (no exposure togriseofulvin), a low dose group (1% griseofulvin, the No ObservableEffect Level and a dose that does not induce elevated levels of liverenzymes in plasma), a medium dose group (2% griseofulvin, a dose inwhich elevated levels of liver enzymes in the plasma begin to bedetected), and a high dose group (5% griseofulvin, a dose that clearlyelicits a toxic effect to the liver, as measured by elevated levels ofliver enzymes in the plasma). Cell proliferation was measured asdescribed, supra. Griseofulvin is recognized as a hepatotoxin, causingliver cell proliferation and porphyria. Griseofulvin was administered tomice in their chow (1% w/w) for 5 days. After 5 days of treatment, cellproliferation in exposed mice showed increased liver cell proliferationat a dose (1%) reported as a No Observable Effect Level (NOEL) (see FIG.6). The 1% dose did not induce elevated levels of plasma liver enzymes,which is consistent with the NOEL (data not shown). The increase inliver cell proliferation at the NOEL dose (i.e., the low dose) was anunanticipated toxic response to griseofulvin since the expected resultwas a lack of observable toxicity based on the NOEL and reports in theliterature.

Example 10 Unanticipated Decrease in Mammary Epithelial CellProliferation in Normal and Ovariectomized Rats after Exposure to LowDoses of Selective Estrogen Receptor Modulators

Rats were maintained in the University of California Berkeley AnimalFacility or the KineMed vivarium in a climate controlled environmentwith a 12 hour light/12 hour dark cycle. Animal care and experimentalprocedures were approved by the University of California, Berkeley,Animal Care and Use Committee or the KineMed's Internal Animal Care andUse Committee depending on the site of the study. Rats were maintainedon standard rodent chow and water provided ad libitum. Rats weresacrificed by CO₂ asphxyiation or anesthetized with isoflurane prior toterminal bleed by heart puncture.

Stable Isotope Labeling.

Rats were labeled with deuterated water (heavy water, ²H₂O) by receivinga bolus i.p. injection of sterile 99.8% ²H₂O with 0.9% NaCl to quicklybring them up to desired body water enrichment quickly followed byreplacement of drinking water with an enriched with ²H₂O to either 4 or8% depending on the study. When 8% was used, injections were done with ½the injection volume twice to avoid injecting to great a volume. The²H₂O used for each study will be indicated in describing the studydesigns (below).

Selective Estrogen Receptor Modulator (SERM) Studies:

The anti-proliferative efficacy of two different SERMS was tested inintact female Sprague-Dawley rats and Sprague-Dawley rats that had beenovariectomized with or without 17 beta-estradiol replacement. Slowrelease pellets were purchased from Innovative Research. The levels ofdrugs chosen were at the very low end of what was previously reported toeffect mammary gland biology and carcinogenesis (5 mg, (Weckbecker G,Tolcsvai L, Stolz B, Pollak M, and Bruns C. Somatostatin analogueoctreotide enhances the antineoplastic effects of tamoxifen andovariectomy on 7,12-dimethylbenz(alpha)anthracene-induced rat mammarycarcinomas. Cancer Res 54: 6334-6337, 1994)). Initially, 2.5, 5, and 10mg tamoxifen pellets (designed to continuously release drug over 21days) were selected in an attempt to produce a dose-response curve. Muchlower doses of tamoxifen, 0.1, 0.5 and 1.0 mg were then investigated.Based on results of the tamoxifen study, raloxifene was alsoinvestigated at the lower doses (0.1-2.5 mg 21 day pellets) thanreported in the literature (s.c. injection dose equivalent to ˜5.9 mg21-day pellet) (Kubatka P, Bojkova B, Kalicka K, Chamilova M, AdamekovaE, Ahlers I, Ahiersova E, and Cermakova M. Preventive effects ofraloxifene and melatonin in N-methyl-N-nitrosourea-induced mammarycarcinogenesis in female rats. Neoplasma 48: 313-319, 2001).

Rats were anesthetized with isoflurane. Pellets of drugs, estradioland/or placebo pellets were aspetically inserted s.c. above the shoulderthrough a small incision (<½ cm). Wound clips were used to close theincisions.

Bone Marrow Isolation:

Femoral bone marrow was flushed out with Medium 199 and collected. Thecells were centrifuged at 100×g to collect the pellet.

Enzymatic Cell Dissociation of Mammary Gland:

MEC were isolated from rat tissue by enzymatic cell dissociation aspreviously described (Yang J, Guzman R, Richards J, Jentoft V, DeVault MR, Wellings S R, and Nandi S. Primary culture of human mammaryepithelial cells embedded in collagen gels. Journal of the NationalCancer Institute 65: 337-343, 1980). In brief, mammary tissue wasenzymatically dissociated to a single cell suspension by digestingovernight at 37° C. in Medium 199 (Invitrogen, Carlsbad, Calif.)containing 0.5% collagenase type IV (Worthington Biochemical, Lakewood,N.J.) and a 1:100 dilution of antibiotic/antimycotic cocktail(Invitrogen, Carlsbad, Calif.). Mammary glands from one animal werefinely sectioned before digesting in a total volume of ˜50 ml. Digestatewas then centrifuged at 300×g for 10 minutes, supernatant removed, andcell pellet resuspended in 2.5 ml of Medium 199 (warmed to 37° C.). Toremove extracellular DNA and reduce cell clumping 100 μl/ml of DNAse I(2,000 Kunitz units/ml) (Sigma-Aldrich, St. Louis, Mo.) was added to thecells in warm medium. Cells were vortexed on highest setting for >1minute, and cell suspension was filtered through 40 micron nylon meshprior to MEC isolation. This step removed extracellular DNA therebypreventing contamination and clumping of cells during subsequent Percollgradient separation or immunomagnetic bead isolation.

Isolation by Percoll Gradient Centrifugation:

Cells were resuspended in 2.5 ml of Medium 199 and layered on top of apreviously prepared Percoll gradient (16 ml of Medium 199 plus 1.2 ml oflox Hanks Balanced Salt Solution (GibcoBRL, Grand Island, N.Y.) plus10.8 ml of Percoll centrifuged at 20,000×g for 1 hour). Centrifugationwas at 800×g for 15 min to achieve cell separation. The middle layer(isolated MEC) was removed and washed once with Medium 199.

MEC isolation by Immunomagnetic Bead Method:

Cells were isolated using an immunomagnetic bead method (MACS™) permanufacturer's recommendations (Miltenyi Biotech Inc., Auburn, Calif.).In brief, cells were pelleted and rinsed twice with 1 ml of labelingbuffer (0.5% bovine serum albumin and 2 mM EDTA). Cells were incubatedin 100 μl of a 1:50 dilution of primary mouse anti-rat epithelialmembrane antigen (EMA) antibody (University of Iowa, DevelopmentalStudies Hybridoma Bank, clone Ha4C19, custom biotinylated by VectorLabs, Burlingame, Calif.) in labeling buffer for 30 minutes at 4° C.Cells were rinsed by adding 1 ml labeling buffer, pelleting at 300×g.For secondary labeling with streptavidin or anti-biotin magnetic beads(Miltenyi Biotech Inc., Auburn, Calif., 20 μl beads per 10⁷ cells in 80μl labeling buffer) cells were incubated in the recommendedconcentration of immunomagnetic reagent in labeling buffer for 30minutes at 4° C. Cells were rinsed using 1 ml labeling buffer, pelletedat 300×g and re-suspended in 500 μl of labeling buffer for loading ontomagnetic columns (Miltenyi Biotech Inc., Auburn, Calif.). Columns wereplace in the magnetic holder and were preconditioned with labelingbuffer, samples were loaded, rinsed 3 times with labeling buffer toelute unlabeled cells. Columns were then removed from the magnet and thepositive cell fraction was plunged off column with 1 ml of labelingbuffer and collected. Cells were rinsed in 1×PBS to remove excess EDTA(EDTA can interfere with DNA hydrolysis enzymes).

DNA Isolation, Hydrolysis to Nucleosides, and Derivatization for GC/MSAnalysis:

The procedures for precipitation of DNA and hydrolysis to nucleosideshave been described in detail previously (McCune J M, Hanley M B, CesarD, Halvorsen R, Hoh R, Schmidt D, Wieder E, Deeks S, Siler S, Neese R,and Hellerstein M. Factors influencing T-cell turnover inHIV-1-seropositive patients [see comments]. Journal of ClinicalInvestigation 105: R1-8, 2000). In brief, cells (MEC and bone marrowcells) were lysed and DNA was isolated using Qiagen QiAmp columns. TheDNA was subjected to enzymatic hydrolysis using nuclease P1 (Roche,Indianapolis, Ind.), snake venom phosphodiesterase I (Sigma, St Louis,Mo.), DNAse (Sigma, St. Louis) and alkaline phosphatase (Sigma, StLouis, Mo.) under basic conditions. Recently, a two enzyme method hasbeen developed that considerably reduces the expense and increases thethroughput. This method involves an acid hydrolysis with acidphosphatase and nuclease S1, both commercially available from a varietyof sources.

Derivatization Methods:

In the initial studies in mice and hormone treated rats, the dA wasconverted to the aldonitriletriacetate (dRATA) derivative as previouslydescribed. The derivatization method has been previously described(Neese R A, Misell L M, Turner 5, Chu A, Kim 3, Cesar D, Hoh R, AnteloF, Strawford A, McCune J M, Christiansen M, and Hellerstein MK.Measurement in vivo of proliferation rates of slow turnover cells by²H₂O labeling of the deoxyribose moiety of DNA. Proc Natl Aced Sci USA99: 15345-15350, 2002.). In addition, a newer more sensitive method ofDNA derivitization was developed and used for subsequent studies of SERMdrug treatment (Raloxifene and Tamoxifen). This pentafluorobenzylderivative (PFBHA) was prepared by reaction of the enzymatic DNA digestwith excess pentafluorobenzyl hydroxylamine under acidic conditions,followed by acetylation with acetic anhydride.

GC/MS Analysis:

An HP model 5971 or 5973 MS with 5890 GC and autosampler(Hewlett-Packard, Palo Alto, Calif.) were used. Abundances of ions atmass to charge ratio (m/z) 198, 199 were quantified for the dRATAderivative, (m/z) 245, 246 were quantified for the PTA derivative and(m/z) 435, 436 were quantified for the PFBHA derivative under selectedion recording mode for derivatized deoxyribose (Neese R A, Siler S Q,Cesar D, Antelo F, Lee D, Misell L, Patel K, Tehrani S, Shah P, andHellerstein M K. Advances in the stable isotope-mass spectrophotometricmeasurement of DNA synthesis and cell proliferation. AnalyticalBiochemistry 298: 189, 2001). Background isotopic enrichment, of DNAstandards ran concurrently with samples, and was subtracted from sampleenrichments. EM+1 (excess M+1 over background enrichment) (m/z 199, 246or 436) enrichments of MEC were divided by bone marrow (a fully-turnedover tissue) EM+1 enrichments from the same animal to determinefractional turnover of MEC (Neese R A, Siler S Q, Cesar D, Antelo F, LeeD, Misell L, Patel K, Tehrani S, Shah P, and Hellerstein M K. Advancesin the stable isotope-mass spectrophotometric measurement of DNAsynthesis and cell proliferation. Analytical Biochemistry 298: 189,2001).

Measurement of Body ²H₂O Enrichments:

Body water enrichments were determined by GC/MS as described previously(Neese R A, Siler S Q, Cesar D, Antelo F, Lee D, Misell L, Patel K,Tehrani S, Shah P, and Hellerstein M K. Advances in the stableisotope-mass spectrophotometric measurement of DNA synthesis and cellproliferation. Analytical Biochemistry 298: 189, 2001).

Calculations:

Enrichments of bone marrow have previously been shown by the inventor'slab to reach asymptotic values within 7-10 days of labeling in rodents(Neese R A, Misell L M, Turner S, Chu A, Kim J, Cesar D, Hoh R, AnteloF, Strawford A, McCune J M, Christiansen M, and Hellerstein M K.Measurement in vivo of proliferation rates of slow turnover cells by²H₂O labeling of the deoxyribose moiety of DNA. Proc Natl Acad Sci USA99: 15345-15350, 2002) and can be used as an essentially completelyturned-over comparison tissue in rats, to determine percent replacementof rat and mouse MEC (Neese R A, Siler S Q, Cesar D, Antelo F, Lee D,Misell L, Patel K, Tehrani S. Shah P, and Hellerstein M K. Advances inthe stable isotope-mass spectrophotometric measurement of DNA synthesisand cell proliferation. Analytical Biochemistry 298: 189, 2001).

The proliferation and replacement rates of MEC after ²H₂O labeling arecalculated based on the precursor-product relationship as described,supra.

${{{Fraction}\mspace{14mu} {of}\mspace{14mu} {new}\mspace{14mu} {{cells}(f)}} = \frac{{EM}_{1}({MEC})}{{EM}_{1}({BM})}},$

where EM₁=excess abundanceIn the M+1 mass isotopomer of derivatized dR, and BM represents bonemarrow cells.

Fractional replacement rate constant(k, day −1)=−ln[l−f]

Half-life(t1/2,d)=0.693÷k

SERM Treatment of Ovariectomized and Intact Rats:

Results for GC/MS analysis of the effects of SERMs on MEC proliferationrates suggest that both tamoxifen and raloxifene are efficaciousanti-proliferative agents at even the lowest dose, a finding that wascompletely unexpected given the lack of any data to suggest that theSERMs would be active at such low doses. In fact, the literature teachesthat much higher doses of SERMs are necessary to see an effect on thereduction in MEC proliferation but our data indicate the contrary aseven extremely low doses have robust effects (see FIG. 7). Significantreductions of MEC proliferation were observed at all doses of tamoxifenand raloxifene when rats were treated with drug and labeled for 7 dayswhether in the presence or absence of estrogen (see FIG. 7).

Example 11 Unanticipated Major Contribution of Glyceroneogenesis inAdipose TG Synthesis After Exposure to Rosiglitazone

Rosiglitazone has been reported to increasephosphoenolpyruvate-carboxykinase (PEPCK) expression and activity inadipocytes (Glorian, M., Duplus, E., Beale, E. G., Scott, D. K.,Granner, D. K., and Forest, C. (2001) Biochimie 83, 933-943; Duplus, E.,Benelli, C., Reis, A. F., Fouque, F., Velho, G., and Forest, C. (2003)Biochimie 85, 1257-1254; Tordjman, J., Khazen, W., Antoine, B., Chauvet,G., Quette, J., Fouque, F., Beale, E. G., Benelli, C., and Forest, C.(2003) Biochimie 85, 1213-1218) and has also been shown to increase thelow levels of glycerol-kinase activity found in adipose tissue (Gunn, H.P., Li, Y., Jensen, M. V., Newgard, C. B., Steppan, C. M., and Lazar, M.A. (2002) Nat Med 8, 1122-1128), which was thought to be the primarymechanism of adipose TG synthesis after exposure to thiazolidinediones.In contrast, based on in published in vitro data, glyceroneogenesis wasthought to play a very minor role in this process. The Applicant hasdiscovered, however, that rodents exposed to the thiazolidinedionerosiglitazone in fact have a marked increase in glyceroneogenesis andthat this increase in glyceroneogenesis is the primary contributingfactor in adipose triglyceride synthesis, which is contrary to thecommon wisdom in the art based on a significant body of in vitro datashowing that glycerol kinase activity is the predominant mechanismunderlying adipose TG synthesis.

Animal Studies:

Mice.

Four-week-old male C57B1/6J mice (16-18 g; Jackson Laboratories, BarHarbor, Me.) were used. Mice were fed ad libitum a high carbohydrate,low fat (HC, 70% carbohydrate, 10% fat) diet or a low carbohydrate, highfat diet (LC, 35% carbohydrate, 45% fat) (Research Diets Inc., NewBrunswick, N.J.). An additional group of mice were fed a HC dietcontaining rosiglitazone (6.34 mg/kcal diet), which resulted in a doseof approximately 3 mg/kg/mouse/day (Research Diets Inc., New Brunswick,N.J.). After 11 days on diet, all animals were given a priming dose of99.8% 2H₂O-saline via intraperitoneal injection to achieve ˜4.8% ²H₂Oenrichment in body water (30 μl/g mouse) followed by administration of8% ²H₂O in drinking water. Mice (n=6 per group) were sacrificed after 15or 64 days on heavy water. Mesenteric, epidydimal, retroperitoneal, andinguinal adipose tissue depots were removed, and blood and urine sampleswere obtained.

Rats.

Sprague-Dawley rats (400-500 g, Charles River Laboratories, Wilmington,Mass.) were purchased with an indwelling carotid artery catheter inplace. Rats were fed ad libitum a Purina chow diet. After an overnightfast, animals were anesthetized with isoflurane and a priming dose of99.8% ²H₂O-saline was given via intraperitoneal injection, as above.After a minimum of 60 minutes, to allow for 2H2O equilibration with bodywater, two baseline blood samples were collected (times −30 and −15min.). At time 0, fructose was infused intravenously (16-19 mg/kg/minfor 4 hours). Blood samples were taken after 3 and 4 hours of fructoseadministration. Animals (n=4) were sacrificed after the 4 hour blooddraw and a final blood sample was collected via cardiac puncture.

Isolation of Acylglyceride-Glycerol from Adipose Tissue:

Lipids from adipose were extracted by a modified Folch extraction(Folch, J., Lees, M., and Sloane Stanley, G. H. (1957) J Biol Chem 226,497-509). The lipid fraction was transesterified by incubation with 3Nmethanolic HCl (Sigma-Aldrich, St. Louis, Mo.) at 55° C. for 60 min.Fatty acid methyl esters were separated from glycerol by the Folchtechnique. The aqueous phase containing glycerol was lyophilized, andglycerol was converted to glycerol triacetate by incubation with aceticanhydride-pyridine (2:1) as described elsewhere (Siler, S. Q., Neese, R.A., Parks, E. J., and Hellerstein, M. K. (1998) J Lipid Res 39,2319-2328).

Isolation of TG-Glycerol from Plasma:

Plasma, obtained from fresh whole blood, was extracted by the Folchtechnique. TG was isolated by TLC as described previously (Jung, H. R.,Turner, S. M., Neese, R. A., Young, S. G., and Hellerstein, M. K. (1999)Biochem J343 Pt 2, 473-478). Glycerol isolation and derivatization werethen performed as described, supra.

Measurements of 2H2O Enrichment in Body Water.

²H₂O enrichment in body water (from plasma or urine) was measured by oneof two methods. Briefly, 15-20 μL of plasma or urine were reacted in anevacuated GC vial with calcium carbide to produce acetylene. Theacetylene gas was then removed with a syringe and injected into a GCvial containing 10% bromine in carbon tetrachloride and incubated atroom temperature for 2 h to produce tetrabromoethane. Excess bromine wasneutralized with 25 μL of 10% cyclohexene, and the sample was suspendedin ethyl acetate (Collins, M. L., Eng, S., Hoh, R., and Hellerstein, M.K. (2003) J Appl Physiol 94, 2203-2211). Alternatively, the acetylenegas was directly measured by a new mass spectrometric method (Previs, S.F., Hazey, J. W., Diraison, F., Beylot, M., David, F., and Brunengraber,H. (1996) J Mass Spectrom 31, 639-642). Briefly, 25 μl of sample wasinjected into a closed Exitainer vial containing calcium carbide in adry helium atmosphere. A small amount (0.5 ml) of the acetylene gasgenerated from the reaction was removed and injected into another closedvial with a helium atmosphere for direct analysis. The two methods, usedwith standard curves, give identical results. However, the directacetylene method is less time consuming, and thus became the preferredmethod during the course of this study.

GC-MS Analyses:

Glycerol-triacetate was analyzed for isotope enrichment by GC-MS asdescribed previously (Slier, S. Q., Neese, R. A., Parks, E. J., andHellerstein, M. K. (1998) J Lipid Res 39, 2319-2328). Mass isotopomerabundances were analyzed by selected ion monitoring of mass-to-chargeratios (m/z) 159-161 (M0-M2). Tetrabromoethane was analyzed for isotopeenrichment by a GC-MS method as described previously (Neese, R. A.,Slier, S. Q., Cesar, D., Antelo, F., Lee, D., Misell, L., Patel, K.,Tehrani, S., Shah, P., and Hellerstein, M. K. (2001) Anal Biochem 298,189-195). A model 6890 GC with 5973 mass spectrometer (AgilentTechnologies, Palo Alto, Calif.) fitted with a DB-225 fused silicacolumn (J&W, Folsom, Calif.) was used in chemical ionization mode. Theisotopic enrichment of acetylene (m/z 26 and 27) was measured bycycloidal mass spectrometry (Monitor Instruments, Pittsburgh, Pa.), andthe percentage of body water enrichment was calculated by comparison toa standard curve prepared gravimetrically from water and ²H₂O.

Calculations

Body ²H₂O Enrichment:

Isotope enrichments of body ²H₂O were determined by comparison withstandard curves using ²H₂O mixed in known proportions with unlabeledwater and conversion to tetrabromoethane, or more recently, acetylene.Mass spectrometric analysis was described previously (Neese, R. A.,Siler, S. Q., Cesar, D., Antelo, F., Lee, D., Misell, L., Patel, K.,Tehrani, S., Shah, P., and Hellerstein, M. K. (2001) Anal Biochem 298,189-195).

[²H]Glycerol Enrichment

Isotope enrichments of [²H]glycerol derived from acylglycerides werecalculated by subtraction of mass isotopomer abundances in unlabeledglycerol standards (Hellerstein, M. K., and Neese, R. A. (1999) Am JPhysiol 276, E1146-1170). EM1 and EM2 were calculated as a fraction ofthe sum of mass isotopomers M0-M2, as previously described for MIDAcalculations (Turner, S. M., Murphy, E. J., Neese, R. A., Antelo, F.,Thomas, T., Agarwal, A., Go, C., and Hellerstein, M. K. (2003) Am JPhysiol Endocrinol Metab 285, E790-803).

MIDA Calculations of n and A^(∞)1:

MIDA is a technique based on combinatorial analysis of the labelingpatterns present in polymers, as described supra and in U.S. Pat. No.5,338,586. Briefly, the EM2 to EM1 ratio (R) is one embodiment of thislabeling pattern. R is dependent on two factors: the proportion (p) oflabeled hydrogen atoms present in tissue water (i.e., the enrichment of²H₂O in tissue water) and the possible number of C—H bonds in glycerolthat are derived from this tissue water (n). If one assumes that the2H-isotopic enrichment (p) of hydrogen in each actively incorporated C—Hof α-GP is equal to the 2H-enrichment of body water and that 2H₂Oenrichment is constant during the labeling period, n can be calculatedfrom the measured p in body water and the measured R.

Using calculation algorithms based on combinatorial probabilities aspreviously described (Turner, S. M., Murphy, E. J., Neese, R. A.,Antelo, F., Thomas, T., Agarwal, A., Go, C., and Hellerstein, M. K.(2003) Am J Physiol Endocrinol Metab 285, E790-803; Hellerstein, M. K.,and Neese, R. A. (1999) Am J Physiol 276, E1146-1170), a “lookup” tablecan be generated, describing R over a given range of values of p fordiscrete values of n (n=3, n=4, n=5). By using the value of p frommeasured body water enrichment, the expected R at each corresponding nis compared to the measured value of R (based on the lookup table, e.g.,p=4.00% yields R=0.121 for n=3, R=0.143 for n=4, R=0.166 for n=5).Because physiologic samples contain a mixture of glyc3.5 and glyc5 (seebelow), we treat n as a non-integral value for this modeling. A linearregression equation is then generated, reflecting the relationshipbetween R and n at the experimentally determined p (the latter based onmeasured ²H₂O enrichment), and the value for n is calculated from themeasured R (e.g., if p=4.00%, the equation for n is n=43.86R−2.29, sofor R=0.152, n=4.38).

Once n is established, one can then calculate A^(∞)1 or the theoreticalasymptotic value for fully labeled TG-glycerol. Accurate determinationof this asymptotic or plateau value is required for the determination offractional synthesis (f) using the precursor-product or rise-to-plateauapproach:

f=EM ₁ /A ^(∞) ₁

where EM₁ represents isotopic enrichment of the mass +1-labeled speciesof glycerol (i.e. the measured abundance in excess of natural abundance)and A^(∞) ₁ represents the asymptotic or plateau value possible for theisotopic enrichment of the mass+1 species of glycerol, calculated from pand a non-integer value of n.

Statistical Analyses.

One-way ANOVA with planned pair-wise comparisons was used with P<0.05 asthe criterion for significance. An independent group t-test and anonparametric Mann-Whitney test with P<0.05 as the criterion forsignificance was used for the fructose infusion comparison.

Results:

The experiment explored the role of glyceroneogenesis in adipose TGsynthesis following PPAR-γ agonist treatment. PPAR-γ agonists, such asrosiglitazone, are insulin sensitizing agents that induce a number ofactions, including stimulation of adipogenesis and increased fatstorage. PPAR-γ is required for the transcription of the PEPCK gene inadipocytes and rosiglitazone has been shown to induce expression ofPEPCK in adipose tissue (Duplus, E., Benelli, C., Reis, A. F., Fouque,F., Velho, G., and Forest, C. (2003) Biochimie 85, 1257-1264).Conversion of oxaloacetate to phosphoenolpyruvate via PEPCK is therate-limiting step in glyceroneogenesis. Rosiglitazone has been shown tosignificantly increase glycerol-kinase activity in isolated adipocytes(Guam, H. P., Li, Y., Jensen, M. V., Newgard, C. B., Steppan, C. M., andLazar, M. A. (2002) Nat Med 8, 1122-1128). A physiologically significantcontribution to adipogenesis from increased glycerol kinase activitywould result in a decrease in n, whereas an increased contribution fromglyceroneogenesis should increase n. The latter was clearly observed(FIG. 8). The observed increase in n with rosiglitazone treatmentprovides in vivo evidence for the significance of up-regulation ofglyceroneogenesis, presumably via increased PEPCK expression, relativeto up-regulation of glycerol kinase. This finding was unexpected asdiscussed, supra.

1-14. (canceled)
 15. A method for high-throughput screening (HTS) ofcombinations or mixtures of two or more compounds for actions onmolecular flux rates in liver collagen synthesis and breakdown, saidmethod comprising: a) administering said two or more compounds to aliving system; b) administering an isotope-labeled substrate to saidliving system for a period of time sufficient for said isotope-labeledsubstrate to enter into and pass through liver collagen synthesis andbreakdown and thereby enter into and label a targeted molecule ormolecules of interest within said liver collagen synthesis and breakdownin said living system, wherein the isotope-labeled substrate is stableisotope-labeled water and the targeted molecule or molecules of interestis selected from the group consisting of proteins, peptides, and aminoacids; c) obtaining one or more samples from said living system, whereinsaid one or more samples comprise one or more isotope-labeled targetedmolecules of interest; d) measuring the isotopic content or the isotopicpattern of the isotope label of the targeted molecule or molecules ofinterest, or the rate of change of the isotopic content or the isotopicpattern of the isotope label of the targeted molecule or molecules ofinterest in the sample; e) calculating molecular flux rates of thetargeted molecule or molecules of interest within said liver collagensynthesis and breakdown; f) measuring the molecular flux rates in saidliver collagen synthesis and breakdown according to steps b) through e)in a living system not administered said two or more compounds; and g)comparing said molecular flux rates in said liver collagen synthesis andbreakdown in said living system administered said two or more compoundsto said molecular flux rates in said liver collagen synthesis andbreakdown in said living system not administered said two or morecompounds to screen said compounds for one or more actions on saidmolecular flux rates.
 16. The method of claim 15, wherein the molecularflux rates in said liver collagen synthesis and breakdown are altered ina living system having a disease of interest relative to a living systemnot having a disease of interest.
 17. The method of claim 16, whereinthe molecular flux rates of said liver collagen synthesis and breakdownof two or more diseases are measured concurrently.
 18. The method ofclaim 17, wherein the concurrent measurement of the molecular flux ratesfrom said liver collagen synthesis and breakdown is achieved by use ofmass spectrometry techniques.
 19. The method of claim 18, wherein theisotope label used is a stable, non-radioactive isotope.
 20. The methodof claim 15, wherein the stable isotope-labeled water is ²H₂O.
 21. Themethod of claim 15, wherein said two or more compounds comprise one ormore known drug agents.
 22. The method of claim 15, wherein said two ormore compounds comprise one or more biological factors, wherein the oneor more biological factors are isolated compounds made by a livingsystem that are administered for screening purposes.
 23. The method ofclaim 15, wherein said actions on molecular flux rates in liver collagensynthesis and breakdown comprise a toxic effect of liver collagensynthesis or liver collagen breakdown.
 24. The method of claim 15,wherein one or more animal models of disease are used for evaluatingsaid one or more actions of said two or more compounds on said molecularflux rates.
 25. The method of claim 24, wherein said one or more animalmodel of disease are selected from the group consisting of hepaticfibrosis, cirrhosis, and hepatocellular necrosis.